Hypoxia/Reoxygenation Effects on Ion Transport across Rat Colonic Epithelium

Ischemia causes severe damage in the gastrointestinal tract. Therefore, it is interesting to study how the barrier and transport functions of intestinal epithelium change under hypoxia and subsequent reoxygenation. For this purpose we simulated hypoxia and reoxygenation on mucosa-submucosa preparations from rat distal colon in Ussing chambers and on isolated crypts. Hypoxia (N2 gassing for 15 min) induced a triphasic change in short-circuit current (Isc): a transient decrease, an increase and finally a long-lasting fall below the initial baseline. During the subsequent reoxygenation phase, Isc slightly rose to values above the initial baseline. Tissue conductance (Gt) showed a biphasic increase during both the hypoxia and the reoxygenation phases. Omission of Cl- or preincubation of the tissue with transport inhibitors revealed that the observed changes in Isc represented changes in Cl- secretion. The radical scavenger trolox C reduced the Isc response during hypoxia, but failed to prevent the rise of Isc during reoxygenation. All changes in Isc were Ca2+-dependent. Fura-2 experiments at loaded isolated colonic crypts revealed a slow increase of the cytosolic Ca2+ concentration during hypoxia and the reoxygenation phase, mainly caused by an influx of extracellular Ca2+. Surprisingly, no changes could be detected in the fluorescence of the superoxide anion-sensitive dye mitosox or the thiol-sensitive dye thiol tracker, suggesting a relative high capacity of the colonic epithelium (with its low O2 partial pressure even under physiological conditions) to deal with enhanced radical production during hypoxia/reoxygenation

Berberine Reduces cAMP-Induced Chloride Secretion in T84 Human Colonic Carcinoma Cells through Inhibition of Basolateral KCNQ1 Channels

Berberine is a plant alkaloid with multiple pharmacological actions, including antidiarrhoeal activity and has been shown to inhibit Cl− secretion in distal colon. The aims of this study were to determine the molecular signaling mechanisms of action of berberine on Cl− secretion and the ion transporter targets. Monolayers of T84 human colonic carcinoma cells grown in permeable supports were placed in Ussing chambers and short-circuit current measured in response to secretagogues and berberine. Whole-cell current recordings were performed in T84 cells using the patch-clamp technique. Berberine decreased forskolin-induced short-circuit current in a concentration-dependent manner (IC50 80 ± 8 μM). In apically permeabilized monolayers and whole-cell current recordings, berberine inhibited a cAMP-dependent and chromanol 293B-sensitive basolateral membrane K+ current by 88%, suggesting inhibition of KCNQ1 K+ channels. Berberine did not affect either apical Cl− conductance or basolateral Na+–K+-ATPase activity. Berberine stimulated p38 MAPK, PKCα and PKA, but had no effect on p42/p44 MAPK and PKCδ. However, berberine pre-treatment prevented stimulation of p42/p44 MAPK by epidermal growth factor. The inhibitory effect of berberine on Cl− secretion was partially blocked by HBDDE (∼65%), an inhibitor of PKCα and to a smaller extent by inhibition of p38 MAPK with SB202190 (∼15%). Berberine treatment induced an increase in association between PKCα and PKA with KCNQ1 and produced phosphorylation of the channel. We conclude that berberine exerts its inhibitory effect on colonic Cl− secretion through inhibition of basolateral KCNQ1 channels responsible for K+ recycling via a PKCα-dependent pathway

Arylthioamides and aryliminothioethers as new slow H2S-releasing agents

Hydrogen sulphide (H2S) is emerging as an important endogenous modulator, which exhibits the beneficial effects of nitric oxide (NO) on the cardiovascular (CV) system, without producing toxic metabolites. H2S exhibits the antioxidant properties of inorganic and organic sulphides, behaving as a scavenger of reactive oxygen species. H2S is biosynthesized in mammalian tissues by cystathionine--synthase (CBS) and cystathionine--lyase (CSE) that is the main source of H2S in the CV. H2S trigger other important effects and the activation of ATP-sensitive potassium channel (KATP) accounts for its vasorelaxing and cardioprotective effects. Furthermore, H2S inhibits smooth muscle proliferation and platelet aggregation. In non-CV systems, H2S regulates the functions of the central nervous system, as well as respiratory, gastroenteric, and endocrine systems. Conversely, H2S deficiency contributes to the pathogenesis of hypertension. Likewise, impairment of H2S biosynthesis is involved in CV complications associated with diabetes mellitus. Many experiments suggest a cross-talk between the H2S and the endothelial NO pathways. In particular, recent observations indicate a possible pathogenic link between deficiencies of H2S activity and the progress of endothelial dysfunction. These biological aspects of endogenous H2S have led several authors to look at this mediator as ‘‘the new NO’’, giving attractive opportunities to develop innovative classes of drugs. NaHS is the prototypical example of H2S-generating agent: it is a rapid H2S-donor and the most widely used H2S-donor for experimental purposes. However, this salt is not suitable for clinical applications, as the quick release of H2S may cause adverse effects, such as a rapid and excessive lowering of blood pressure. For a safer and effective pharmacological administration, ideal H2S-donors should generate H2S with slower releasing rates. Organic polysulphides of garlic, such as diallyl disulphide (DADS), act as H2S-releasing compounds, with a relatively slow mechanism that requires the presence of reduced glutathione. Other examples of original synthetic H2S-releasing agents have been described in the literature, including a number of aminothiol derivatives, and the phosphinodithioate derivative GYY4137. Furthermore, some H2S-releasing chemical moieties, such as the dithiolethione, the thioamide and the isothiocyanate, have been used for synthesizing multifunctional drugs. In this study, we report the synthesis and pharmacological evaluation of a series of new arylthioamide and aryliminothioether as “smart” H2S-releasing drugs

Hypoxia attenuate ionic transport in the isolated gill epithelium of Carcinus maenas

The gills are osmorespiratory organs of aquatic organisms and the prime target of environmentally induced hypoxia. We have studied the impact of severe hypoxia (0.5 mg O2/L) on the ionic transport across posterior gills of Carcinus maenas acclimated to 12 ppt seawater (DSW). The short-circuit current (Isc) across hemilamellae from gills i.e. active ion transport was studied in micro Ussing chambers. Hypoxia induced by deoxygenation of the basolateral side, and not the apical side, resulted in time-dependent inhibition of Isc and full recovery of Isc after reoxygenation. Exposure of the crabs to severe 7 h hypoxia decreased the specific activity of Na+, K+-ATPase in the gills by 36%. Full recovery of enzyme activity occurred in fasted crabs after 3 days of reoxygenation. The intensity of Western blotting bands was not different in the gills of oxygenated, hypoxic and reoxygenated crabs. The reversible, nonspecific blocker of K+ channels Cs and hypoxia inhibited over 90% of Isc which is after reoxygenation fully recovered. The specific blocker of Cl− channels NPPB [5-nitro-2-(3-phenylpropylamino) benzoic acid] blocked Isc by 68.5%. Only the rest of not inhibited Isc in aerated saline was blocked by hypoxia and recovered after reoxygenation. The activity of the antioxidant enzyme catalase was not changed during hypoxia and reoxygenation kept the high enzyme activity in the gills at the level of crabs acclimated to DSW. As a response to hypoxia the presence of 2 mM H2O2 induce an initial slight transient decrease of Isc followed by a rise and after reoxygenation fully recovered Isc. Incubation of hemilamellae with the antioxidant derivative Trolox did not affect the inhibition of Isc by hypoxia

Gaseous mediators nitric oxide and hydrogen sulfide in the mechanism of gastrointestinal integrity, protection and ulcer healing

Nitric oxide (NO) and hydrogen sulfide (H2S) are known as biological messengers; they play an important role in human organism and contribute to many physiological and pathophysiological processes. NO is produced from l-arginine by constitutive NO synthase (NOS) and inducible NOS enzymatic pathways. This gaseous mediator inhibits platelet aggregation, leukocyte adhesion and contributes to the vessel homeostasis. NO is known as a vasodilatory molecule involved in control of the gastric blood flow (GBF) and the maintenance of gastric mucosal barrier integrity in either healthy gastric mucosa or that damaged by strong irritants. Biosynthesis of H2S in mammals depends upon two enzymes cystathionine-β-synthase and cystathionine γ-lyase. This gaseous mediator, similarly to NO and carbon monoxide, is involved in neuromodulation, vascular contractility and anti-inflammatory activities. For decades, H2S has been known to inhibit cytochrome c oxidase and reduce cell energy production. Nowadays it is generally considered to act through vascular smooth muscle ATP-dependent K+ channels, interacting with intracellular transcription factors and promote sulfhydration of protein cysteine moieties within the cell, but the mechanism of potential gastroprotective and ulcer healing properties of H2S has not been fully explained. The aim of this review is to compare current results of the studies concerning the role of H2S and NO in gastric mucosa protection and outline areas that may pose new opportunities for further development of novel therapeutic targets

The impact of host-microbe interactions on murine colonic secretomotor function

The overall objective of this thesis was to gain further insight into the mechanisms underlying commensal microbial influences on intestinal ion transport. In this regard, I examined the impact of commensal host-microbe interactions on colonic secretomotor function in mouse. I first examined the influence of two different probiotic (microorganisms which, when given in adequate amounts, can confer health benefits upon the host) strains, Bifidobacterium infantis 35624 and L. salivarius UCC118 on active colonic ion transport in the mouse, using the Ussing Chamber. I found that both probiotics appear to have converging effects on ion transport at a functional level. However, L. salivarius UCC118 may preferentially inhibit neurally-evoked ion transport. Next I examined the impact of the host microbiota itself on both baseline and stimulated colonic secretomotor function as well as probiotic induced changes in ion transport. I provide further evidence that the microbiota is capable of mediating alterations in colonic ion transport, and specifically suggests that it can influence cAMP-mediated responses. Finally, it has been well documented that many probiotics elicit their effects via secreted bioactives, therefore, I studied the effects of microbially produced GABA, contained in supernatants from the commensal microbe Lactobacillus brevis DPC6108, on colonic secretomotor function. In conclusion, I believe that commensal microbes have an important and strain specific functional influence on colonic ion transport and secretomotor function and these effects can be mediated via extracellular bioactives. Moreover, I believe that functional ex-vivo studies such as those carried out in this thesis have a critical role to play in our future understanding of host-microbe interactions in the gut

Role of Aquaporins in Breast Cancer Progression and Metastasis

There are various limitations regarding the current pharmacological options for the treatment of breast cancer in terms of efficacy, target selectivity, side effect profile and survival. Endocrine-based therapy for hormone-sensitive cancers such as that of the breast is one of the most effective and well-tolerated therapeutic options but is hampered by either intrinsic or acquired resistance, resulting in a more aggressive form of the disease. It is generally agreed that this process occurs in parallel with cellular transition from epithelial to mesenchymal phenotype (EMT), with consequent enhancement of proliferative capacity, migrative ability and invasive potential. Aquaporins (AQPs) represent a large family of water channel proteins which are widely distributed in various tissues and which play a role in the physiological maintenance of the extracellular environment particularly to regulate electrolyte-water balance. Accumulating evidence shows that expression of several AQPs is modulated in cancer tissues, and this correlates with tumor grade. AQPs 1 and 3–5 are also involved in breast cancer invasion, through modulating the activity of various growth factors, signaling molecules and proteolytic enzymes. We review current data on the involvement of these proteins in processes associated with malignant progression and discuss possible applications of AQP-based therapy as an effective means of inhibiting cancer cells from metastasizing

Inactivation of pathogens on food and contact surfaces using ozone as a biocidal agent

This study focuses on the inactivation of a range of food borne pathogens using ozone as a biocidal agent. Experiments were carried out using Campylobacter jejuni, E. coli and Salmonella enteritidis in which population size effects and different treatment temperatures were investigate