Sa1682 Mechanosensory Signaling in Human Enteric Glial Cells

Table 1. Fold mRNA upregulation by LPS (200µg/ml) + IFN g (10µg/ml) of selected genes

AGA Abstracts

Ret+ myenteric neurons in the ileum and 19 ± 3% in the colon express calbindin. In contrast to iMNs, Ret expression in calbindin+ myenteric neurons is restricted to a subset. These data show that Ret expression is maintained within specific subsets of enteric neurons and that diminished Ret function is associated with a sex-dependent defect in GI motility. Altering Ret expression or function may thus offer a sexually dimorphic therapeutic strategy for GI motility disorders.

Sa1682 Mechanosensory Signaling in Human Enteric Glial Cells Fernando Ochoa-Cortes, Andromeda Linan Rico, Alix Zuleta-Alarcon, Min Zhou, Emmett Whitaker, Iveta Grants, Violeta N. Mutafova-Yambolieva, Leonie Durnin, Bradley J. Needleman, Alan Harzman, Rosario Cuomo, Fabio Turco, Fievos L. Christofi Background: Clinical observations, animal or human tissue studies implicate enteric glial cells (EGC) in modulation of motility, purinergic signaling, GI disorders and inflammatory diseases. Human EGC (hEGC) are sensitive to mechanical stimulation (MS), suggesting that movement (i.e. peristaltic activity) can activate them. This study focused on mechanosensory signaling in hEGC and whether different modes of MS can influence Ca2+signaling, electrophysiological behavior and purine release. Experimental Design: Cultures of purified myenteric hEGC were from 12 GI-surgical specimens (colon/jejunum); s100B-immunoreactivity occurred in>95% cells. MS was used to test its effect on Ca2+transients, Ca2+oscillations and Ca2+waves in networks of hEGC. Pressure ‘puff' application of buffer (or drug/1mM) onto a single cell or touch/compression was used to evoke responses. Increase in pulsatile flow or radial stretch was used to induce a population response. Patch-clamp (current-clamp mode) was used to monitor Vm membrane potential. Rotational shaking (belly-dancer motion) was used to provoke release of ATP. Real-time fluo-4/Ca2+imaging was done at 10-30 frames/ sec on an upright Eclipse FN1 Nikon System equipped with an Andor iXon Ultra-EMCCD camera. Results: MS of a single cell elicited propagating Ca2+waves. Waves induced by MS had the slowest propagation velocity (20µm/sec) when compared to Ca2+waves triggered by purines (ATP/UTP) or a neurotransmitter (5-HT, 1mM, 150µm/sec), and the latter had the fastest velocity (5-HT >>ATP/UTP> MS, p<0.001). Radial-stretch caused Ca2+transients in hEGC (84 cells analyzed). A 1% prestretch (<1µm stretch) caused Ca2+transients (26% cells) and releasing the stretch caused an off-response (32% cells). Additional stretch of 3%9% (2-5µm/10-48kPa) induced a delayed Ca2+response (or oscillations) after stretch in 52% of cells analyzed (44/88 cells). MS, by increasing pulsatile flow, triggered rhythmic Ca2+oscillations. Touch/compression (4/4 cells) or pressure puff (9/12 cells) onto a single cell evoked a reversible Vm-depolarization (6/8 SMP, 3/4 MP). Pressure puff on a cell located 1000µm away from the patch-recording electrode evoked a Vm-depolarization (~4mV). MS evokes purine release in other cells. Basal purine release from hEGC included ATP, ADP, NAD, AMP, ADO and ADPR. Undulating rotational shaking (~100rpms) increased basal release of ATP (1.27±0.14nM) by 1.8 fold (to 1.89±0.22nM, p=0.029, n=12). Conclusions: MS is an important mechanism in activation of hEGC Ca2+signaling (Ca2+oscillations/waves) and purine release. Disruption of Ca2+signaling in mouse EGC is linked to attenuation of motility. We propose MS is an important sensory control in hEGC as a direct feedback mechanism on the ‘motor status of the gut' to provide moment by moment fine-tune modulation during peristaltic activity (DK093499; DK41315;COFIN 2009HLNNRL).

p<0.0001, for each gene of 107 genes analyzed by nanostring; N=4 surgical specimens (4 human subjects), 16 samples for LPS treatment & 16 samples for control

Sa1684 Quantification of the Anisotropic Ratio of Gastric Tissue Conductivities: Methods and Application Peng Du, Niranchan Paskaranandavadivel, Shameer Sathar, Timothy R. Angeli, Gregory O'Grady, Leo K. Cheng Background: Gastric slow wave propagation is coordinated by coupled networks of interstitial cells of Cajal (ICC), including cellular distributions in the longitudinal muscle (LM) and circumferential muscle (CM) layers of the stomach. Extracellular high-resolution (HR) electrical mapping studies have shown that the ratio of slow wave propagation in each direction, i.e., anisotropic ratio (AR), is critical to the maintenance of normal conduction and pacemaker activity. A major component contributing to the AR of active slow wave propagation is the AR of passive tissue conductivities. Therefore, an in-vivo method for quantifying AR of gastric tissue conductivities is needed. Aim: The aim of this study was to determine the AR of passive gastric conductivity. We hypothesized that passive response of gastric tissue to an extracellular stimulus can be used to determine the AR. Method: HR mapping using flexibleprinted-circuit electrode array (16x16; 4 mm spacing) was conducted in cross-breed weaner pigs, with the electrodes positioned on the serosa of the corpus. A pair of stimulation electrodes was inserted through the center of the electrode array into the gastric musculature. The following unipolar stimulation was applied: 2-10 mA amplitude in consecutive 2 mA increments; 20 s period; 0.2 s pulse-width. Recordings of the extracellular potentials due to pacing were obtained at 512 Hz. Amplitudes of extracellular potentials were calculated by measuring the peak potential response due to pacing at each electrode. An amplitude map was reconstructed by arranging the amplitudes in the same configuration as the electrode array. A tissue conductivity model with two parameters presenting the CM and LM conductivities were fitted to the amplitude map of experimental recordings. The root-mean-squared (RMS) error between the output of the model and experimental recordings was minimized to identify the conductivities. Values are expressed as mean±SEM. Results: Passive extracellular potentials due to stimuli were reliably detected in all of 15 recordings. Potentials decayed as distance increased from the stimulus site, and were correlated to the amplitudes of the stimuli (R2: 0.99±0.01). The average fitted conductivity was highly anisotropic, being 14.5±2.1 mS/mm in the CM direction and 4.8±0.3 mS/mm in the LM direction. The average of the calculated AR was 2.5±0.3 and showed no statistical difference between the stimulus amplitudes (Student's t-test P-value>0.16 for all). The average of RMS error was 3.4±0.5 mV. Conclusions: AR of passive gastric conductivities were reliably quantified from the spatial decay of potential due to stimulus. These data provide a more complete description of gastric conductivity to inform future physiological studies to detect potential changes in conductivity due to the remodelling of ICC and gastric smooth muscles in diseases.

Sa1683 Molecular Signaling and Dysfunction of the Human Reactive Enteric Glial Cell Phenotype: Implications for Intestinal Infections, Motility and IBD Andromeda Linan Rico, Fabio Turco, Fernando Ochoa-Cortes, Alan Harzman, Razvan Arsenescu, Mahmoud Abdel-Rasoul, Paolo Fadda, Iveta Grants, Rosario Cuomo, Fievos L. Christofi Background: Clinical observations and animal studies implicate enteric glial cells (EGC) in purinergic signaling, modulation of motility, GI disorders and inflammatory diseases. Mechanisms underlying glial responses to inflammation in human GI tract are not understood. Our goal was to identify the ‘reactive human EGC phenotype' (rhEGC) induced by inflammation and probe its functional relevance. Experimental Design: Purified-Human EGC in culture (Gut, 2014) from 15 GI-surgical specimens (mainly from the myenteric plexus of the colon and small-bowel) were used to study gene expression, Ca2+signaling, purinergic and flow-dependent Ca2+signaling by fluo-4/Ca2+ imaging, and ATP release. A nanostring panel of 107 genes was designed as a read out of inflammation, transcription, purinergic signaling, vesicular transport-protein, channels, free radical pathways, and second messenger systems (p<0.001). Cells were activated by bacterial lipopolysaccharide (LPS+IFN g) to induce a rhEGC phenotype. Results: The mRNA gene expression in the rhEGC phenotype induced by LPS was altered in 60% of genes analyzed from a 107 gene panel, including inflammatory genes (54%), purinergic receptor genes (52%), purinergic enzyme genes (46%), channels (40%), vesicular transport genes (50%), transcription factors (88%), junction/barrier proteins (20%), and free radical/other pathway genes (Table 1). The mRNA expression of 95% of affected genes was up-regulated and only 3 genes were down-regulated by LPS (PRKACA, IgFBP5, ADIPOR1). Alterations in purine gene-expression indicated a switch from ATP signaling to ADP/adenosine/UTP signaling. ATP-induced Ca2+ responses were disrupted and ATP release increased 5-fold, whereas s100 b release decreased in response to treatment. Mechanical/pulsatile flow-dependent Ca2+responses were disrupted by treatment, as was store-operated-Ca2+ entry. LPS did not affect cell viability using ENZO-Nuclear-ID Bluegreen cell-viability reagent (p>0.05). Conclusions: Findings suggest a detrimental ‘rhEGC phenotype' operating in the hENS in response to LPS (or infection). The rhEGC phenotype is identified by a complex cascade of pro-inflammatory pathways activated by LPS through Toll-like receptors, leading to alterations of important molecular signaling pathways that include purinergic pathways (receptors/enzymes), second messenger systems, free radical pathways (i.e. huge up-regulation of SOD2), neurotransmitter signaling, TRP channels (TRPA1/TRPV1, sensory signaling), vesicular release proteins (SYT2, SYP, SNAP25, glial transmission), and junction/barrier proteins (CLDN1). Altogether, these molecular changes cause disruption of Ca2+signaling, purinergic signaling and mechanosensory signaling in human enteric glial cells. Findings may have important implications for GI infection, motility, IBD, post-operative ileus and GI disorders (DK093499; COFIN/2009HLNNRL).

Sa1685 LPS Myenteric Neurotoxic Action Is Initiated by Palmitate Through TLR4 Activation Francois Reichardt, Anitha Mallapa, Behtash Nezami, Shanthi Srinivasan Introduction: High-fat diets (HFD) lead to altered gastrointestinal motility and nitrergic myenteric neuronal loss that affects particularly the colon where the enteric TLR4 expression is enhanced. This receptor recognize bacterial lipopolysaccharide (LPS) produced during HFD-induced gut microbiota dysbiosis, but also saturated fatty acids as palmitate that is abundant in HFD. Therefore, we investigated in vitro the possible synergic role of palmitate in the LPS toxic action on enteric cultured neurons, using LPS concentrations established

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AGA Abstracts