Imaging of Nitric Oxide in Nitrergic Neuromuscular Neurotransmission in the Gut

Background: Numerous functional studies have shown that nitrergic neurotransmission plays a central role in peristalsis and sphincter relaxation throughout the gut and impaired nitrergic neurotransmission has been implicated in clinical disorders of all parts of the gut. However, the role of nitric oxide (NO) as a neurotransmitter continues to be controversial because: 1) the cellular site of production during neurotransmission is not well established; 2) NO may interacts with other inhibitory neurotransmitter candidates, making it difficult to understand its precise role. Methodology/Principal Findings: Imaging NO can help resolve many of the controversies regarding the role of NO in nitrergic neurotransmission. Imaging of NO and its cellular site of production is now possible. NO forms quantifiable fluorescent compound with diaminofluorescein (DAF) and allows imaging of NO with good specificity and sensitivity in living cells. In this report we describe visualization and regulation of NO and calcium ($Ca^{2+}$) in the myenteric nerve varicosities during neurotransmission using multiphoton microscopy. Our results in mice gastric muscle strips provide visual proof that NO is produced de novo in the nitrergic nerve varicosities upon nonadrenergic noncholinergic (NANC) nerve stimulation. These studies show that NO is a neurotransmitter rather than a mediator. Changes in NO production in response to various pharmacological treatments correlated well with changes in slow inhibitory junction potential of smooth muscles. Conclusions/Significance: Dual imaging and electrophysiologic studies provide visual proof that during nitrergic neurotransmission NO is produced in the nerve terminals. Such studies may help define whether NO production or its signaling pathway is responsible for impaired nitrergic neurotransmission in pathological states

The role of gut-liver axis in the restriction of intrauterine growth in a model of experimental gastroschisis

PURPOSE: To evaluate the intrauterine growth restriction (IUGR) by the expression of IR-&#946;, IRS-1, IRS-2, IGF-IR&#946; and Ikappa&#946; in experimental model of gastroschisis. METHODS: Pregnant rats at 18.5 days of gestation were submitted to surgery to create experimental fetal gastroschisis (term = 22 days) were divided in three groups: gastroschisis (G), control (C) and sham (S). Fetuses were evaluated for body weight (BW), intestinal (IW), liver (LW) and their relations IW/BW and LW/BW. IR-&#946; and IGF-IR&#946; receptors, IRS-1 and IRS-2 substrates and Ikappa&#946; protein were analyzed by western blotting. RESULTS: BW was lower in G, the IW and IW / BW were greater than C and S (p<0.05) groups. The liver showed no differences between groups. In fetuses with gastroschisis, compared with control fetuses, the expression of IGF-IR&#946; (p<0.001) and Ikappa&#946; (p<0.001) increased in the liver and intestine, as well as IR-&#946; (p<0.001) which decreased in both. In contrast to the intestine, IRS-1 (p<0.001) increased in the liver and IRS-2 decreased (p<0.01). CONCLUSION: The axis of the intestine liver has an important role in inflammation, with consequent changes in the metabolic pathway of glucose can contribute to the IUGR in fetuses with gastroschisis

Dietary antioxidants protect gut epithelial cells from oxidant-induced apoptosis

BACKGROUND: The potential of ascorbic acid and two botanical decoctions, green tea and cat's claw, to limit cell death in response to oxidants were evaluated in vitro. METHODS: Cultured human gastric epithelial cells (AGS) or murine small intestinal epithelial cells (IEC-18) were exposed to oxidants – DPPH (3 μM), H(2)O(2) (50 μM), peroxynitrite (300 μM) – followed by incubation for 24 hours, with antioxidants (10 μg/ml) administered as a 1 hour pretreatment. Cell number (MTT assay) and death via apoptosis or necrosis (ELISA, LDH release) was determined. The direct interactions between antioxidants and DPPH (100 μM) or H(2)O(2) (50 μM) were evaluated by spectroscopy. RESULTS: The decoctions did not interact with H(2)O(2), but quenched DPPH although less effectively than vitamin C. In contrast, vitamin C was significantly less effective in protecting human gastric epithelial cells (AGS) from apoptosis induced by DPPH, peroxynitrite and H(2)O(2) (P < 0.001). Green tea and cat's claw were equally protective against peroxynitrite and H(2)O(2), but green tea was more effective than cat's claw in reducing DPPH-induced apoptosis (P < 0.01). Necrotic cell death was marginally evident at these low concentrations of peroxynitrite and H(2)O(2), and was attenuated both by cat's claw and green tea (P < 0.01). In IEC-18 cells, all antioxidants were equally effective as anti-apoptotic agents. CONCLUSIONS: These results indicate that dietary antioxidants can limit epithelial cell death in response to oxidant stress. In the case of green tea and cat's claw, the cytoprotective response exceed their inherent ability to interact with the injurious oxidant, suggestive of actions on intracellular pathways regulating cell death

In vitro Induction of Entamoeba histolytica Cyst-like Structures from Trophozoites

Inhibition of encystment can be conceived as a potentially useful mechanism to block the transmission of Entamoeba histolytica under natural conditions. Unfortunately, amoeba encystment has not been achieved in vitro and drugs inhibiting the formation of cysts are not available. Luminal conditions inducing encystment in vivo are also unknown, but cellular stress such as exposure to reactive oxygen species from immune cells or intestinal microbiota could be involved. A role for certain divalent cations as cofactors of enzymes involved in excystment has also been described. In this study, we show that trophozoite cultures, treated with hydrogen peroxide in the presence of trace amounts of several cations, transform into small-sized spherical and refringent structures that exhibit resistance to different detergents. Ultrastructural analysis under scanning and transmission electron microscopy revealed multinucleated structures (some with four nuclei) with smooth, thick membranes and multiple vacuoles. Staining with calcofluor white, as well as an ELISA binding assay using wheat germ agglutinin, demonstrated the presence of polymers of N-acetylglucosamine (chitin), which is the primary component of the natural cyst walls. Over-expression of glucosamine 6-phosphate isomerase, likely to be the rate-limiting enzyme in the chitin synthesis pathway, was also confirmed by RT-PCR. These results suggest that E. histolytica trophozoites activated encystment pathways when exposed to our treatment

Oxidative Stress and Mitochondrial Functions in the Intestinal Caco-2/15 Cell Line

Although mitochondrial dysfunction and oxidative stress are central mechanisms in various pathological conditions, they have not been extensively studied in the gastrointestinal tract, which is known to be constantly exposed to luminal oxidants from ingested foods. Key among these is the simultaneous consumption of iron salts and ascorbic acid, which can cause oxidative damage to biomolecules.The objective of the present work was to evaluate how iron-ascorbate (FE/ASC)-mediated lipid peroxidation affects mitochondrion functioning in Caco-2/15 cells. Our results show that treatment of Caco-2/15 cells with FE/ASC (0.2 mM/2 mM) (1) increased malondialdehyde levels assessed by HPLC; (2) reduced ATP production noted by luminescence assay; (3) provoked dysregulation of mitochondrial calcium homeostasis as evidenced by confocal fluorescence microscopy; (4) upregulated the protein expression of cytochrome C and apoptotic inducing factor, indicating exaggerated apoptosis; (5) affected mitochondrial respiratory chain complexes I, II, III and IV; (6) elicited mtDNA lesions as illustrated by the raised levels of 8-OHdG; (7) lowered DNA glycosylase, one of the first lines of defense against 8-OHdG mutagenicity; and (8) altered the gene expression and protein mass of mitochondrial transcription factors (mtTFA, mtTFB1, mtTFB2) without any effects on RNA Polymerase. The presence of the powerful antioxidant BHT (50 microM) prevented the occurrence of oxidative stress and most of the mitochondrial abnormalities.Collectively, our findings indicate that acute exposure of Caco-2/15 cells to FE/ASC-catalyzed peroxidation produces harmful effects on mitochondrial functions and DNA integrity, which are abrogated by the powerful exogenous BHT antioxidant. Functional derangements of mitochondria may have implications in oxidative stress-related disorders such as inflammatory bowel diseases

Computational Insights on the Competing Effects of Nitric Oxide in Regulating Apoptosis

Despite the establishment of the important role of nitric oxide (NO) on apoptosis, a molecular- level understanding of the origin of its dichotomous pro- and anti-apoptotic effects has been elusive. We propose a new mathematical model for simulating the effects of nitric oxide (NO) on apoptosis. The new model integrates mitochondria-dependent apoptotic pathways with NO-related reactions, to gain insights into the regulatory effect of the reactive NO species N2O3, non-heme iron nitrosyl species (FeLnNO), and peroxynitrite (ONOO−). The biochemical pathways of apoptosis coupled with NO-related reactions are described by ordinary differential equations using mass-action kinetics. In the absence of NO, the model predicts either cell survival or apoptosis (a bistable behavior) with shifts in the onset time of apoptotic response depending on the strength of extracellular stimuli. Computations demonstrate that the relative concentrations of anti- and pro-apoptotic reactive NO species, and their interplay with glutathione, determine the net anti- or pro-apoptotic effects at long time points. Interestingly, transient effects on apoptosis are also observed in these simulations, the duration of which may reach up to hours, despite the eventual convergence to an anti-apoptotic state. Our computations point to the importance of precise timing of NO production and external stimulation in determining the eventual pro- or anti-apoptotic role of NO