The enteric nervous system : new developments and emerging concepts

The enteric nervous system (ENS) is an integrative neuronal network, organized in two ganglionated plexuses, myenteric and submucosal, composed of neurons and enteric glial cells, controlling the activity of the smooth muscle of the gut, mucosal secretion and blood flow. The ENS contains as many neurons as the spinal cord, and the functional and chemical diversity of enteric neurons closely resembles that of the central nervous system. This highly integrated neural system is also referred to as the ‘brain-in-the-gut’, because of its capability to function in the absence of nerve inputs from the central nervous system.peer-reviewe

The enteric nervous system : new developments and emerging concepts

The enteric nervous system (ENS) is an integrative neuronal network, organized in two ganglionated plexuses, myenteric and submucosal, composed of neurons and enteric glial cells, controlling the activity of the smooth muscle of the gut, mucosal secretion and blood flow. The ENS contains as many neurons as the spinal cord, and the functional and chemical diversity of enteric neurons closely resembles that of the central nervous system. This highly integrated neural system is also referred to as the ‘brain-in-the-gut’, because of its capability to function in the absence of nerve inputs from the central nervous system.peer-reviewe

Angiotensin II contractile effects in mouse colon: role for pre- and post-junctional AT1A receptors

Aim: This study investigates whether a local renin–angiotensin system (RAS) exists in mouse colon and whether angiotensin II (Ang II) may play a role in the regulation of the contractile activity. Methods: Isometric recordings were performed in vitro on the longitudinal muscle of mouse proximal and distal colon. Transcripts encoding for RAS components were investigated by RT-PCR. Results: Ang II caused, in both preparations, a concentration-dependent contractile effect, antagonized by losartan, AT1 receptor antagonist, but not by PD123319, AT2 receptor antagonist. The combination of losartan plus PD123319 caused no change on the Ang II-induced contraction than losartan alone. Tetrodotoxin, neural blocker, reduced the contractile response to Ang II in the proximal colon, whilst the response was abolished in the distal colon. In both preparations, atropine, muscarinic receptor antagonist, or SR140333, NK1 receptor antagonist, reduced the Ang II responses. Ondansetron, 5-HT3 receptor antagonist, SR48968, NK2 receptor antagonist, or hexamethonium, nicotinic receptor antagonist, were ineffective. The joint application of atropine and SR140333 produced no additive effect. Atropine reduced NK1-induced contraction. Transcripts encoding RAS components were detected in the colon samples. However, just AT1A mRNA was expressed in both preparations, and AT2 mRNA was expressed only in the distal colon. Conclusion: In the murine colon, local RAS may play a significant role in the control of contractile activity. Ang II positively modulates the spontaneous contractile activity via activation of post-junctional and pre-junctional AT1A receptors, the latter located on the enteric neurones, modulating the release of tachykinins and acetylcholine

Angiotensin II positively modulates the spontaneous contractile activity of mouse and human colon via activation of AT1 receptors.

Objective: Angiotensin II (Ang II) is a potent smooth muscle contractile neurohumoral agonist but has not been much investigated with regard to gastrointestinal motor activity. Ang II effects are mediated by specific receptors, the Ang II type 1 (AT1) and the Ang II type 2 (AT2) receptors, which are well expressed in the gut. In this study we evaluated the effects of Ang II on the contractile activity of longitudinal muscle from mouse and human colon and we analysed the subtype(s) of receptors involved in the observed effects. Methods: Mechanical responses to Ang II, in the absence or in the presence of different drugs, were assessed in vitro in colonic longitudinal muscle from mice and humans, as changes in isometric tension. Results: In the murine proximal and distal colon Ang II induced a concentration-dependent muscular contraction, which was reduced by the AT1 receptor antagonist, losartan, but it was not affected by the AT2 receptor antagonist, PD123319. Pretreatment with TTX, sodium voltage-gated neural channel blocker, partially reduced the contractile response to Ang II in the proximal colon, while abolished it in the distal colon. Atropine, muscarinic receptor antagonist, or SR140333, NK1 receptor antagonist, reduced the TTX-sensitive excitatory effects induced by Ang II in both preparations. On the contrary, hexamethonium, nicotinic receptor antagonists, ondansetron, 5-HT3 receptor antagonist, or SR48968, NK2 receptor antagonist, were ineffective. The contraction induced by a selective NK1 receptor agonist was reduced by atropine, whilst SR140333 did not affected carbachol inducing muscular contraction. Ang II induced a muscular contraction even in the human distal colonic longitudinal muscle preparations. The concentration–response curve was shifted to the right by losartan but it was unaffected by PD123319. TTX and atropine partially antagonized the response to Ang II. Conclusion: In the longitudinal muscle preparations from mouse and human colon Ang II positively modulates the spontaneous contractile activity via activation of post-junctional and pre-junctional AT1 receptors, the latter located on the enteric nerves and modulating the release of tachykinins and acethylcoline. In mouse tachykinergic neurons and cholinergic neurons are sequentially recruited by Ang II to induce muscular contraction

Arginine vasopressin, via activation of post-junctional V1 receptors, induces contractile effects in mouse distal colon

The aim of this study was to analyze whether arginine vasopressin (AVP) may be considered a modulator of intestinal motility. In this view, we evaluated, in vitro, the effects induced by exogenous administration of AVP on the contractility of mouse distal colon, the subtype(s) of receptor(s) activated and the action mechanism. Isometric recordings were performed on longitudinal and circular muscle strips of mouse distal colon. AVP (0.001 nM–100 nM) caused concentration-dependent contractile effects only on the longitudinal muscle, antagonized by the V1 receptor antagonist, V-1880. AVP-induced effect was not modified by tetrodotoxin, atropine and indomethacin. Contractile response to AVP was reduced in Ca2+-free solution or in the presence of nifedipine, and it was abolished by depletion of calcium intracellular stores after repetitive addition of carbachol in calcium-freemediumwith addition of cyclopiazonic acid.U-73122, an inhibitor of the phospholipase C, effectively antagonized AVP effects, whilst it was not affected by an adenylyl cyclase inhibitor. Oxytocin induced an excitatory effect in the longitudinalmuscle of distal colon at very high concentrations, effect antagonized by V-1880. The results of this study shown that AVP, via activation of V1 receptors, is able to modulate positively contractile activity of longitudinal muscle of mouse distal colon, independently by enteric nerve activation and prostaglandin synthesis. Contractile response is achieved by increase in cytoplasmatic Ca2+ concentration via extracellular Ca2+ influx from L-type Ca2+ channels and via Ca2+ release from intracellular stores through phospholipase C pathway. No modulation has been observed on the contractility of the circular muscle

Aging modifies receptor expression but not muscular contractile response to angiotensin II in rat jejunum

: The involvement of renin-angiotensin system in the modulation of gut motility and age-related changes in mRNA expression of angiotensin (Ang II) receptors (ATR) are well accepted. We aimed to characterize, in vitro, the contractile responses induced by Ang II, in jejunum from young (3-6 weeks old) and old rats (≥ 1 year old), to evaluate possible functional differences associated to changes in receptor expression. Mechanical responses to Ang II were examined in vitro as changes in isometric tension. ATR expression was assessed by qRT-PCR. Ang II induced a contractile effect, antagonized by losartan, AT1R antagonist, and increased by PD123319, AT2R antagonist, as well by neural blocker ω-conotoxin and by nitric oxide (NO) synthase inhibitor. No difference in the response was observed between young and old groups. AT1 receptor-mediated contractile response was decreased by U-73122, phospholipase C (PLC) inhibitor; or 2-aminoethoxy-diphenylborate (2-APB), inositol triphosphate (IP3) receptor inhibitor; or nifedipine, L-type calcium channel blocker. Age-related changes in the expression of both AT1 receptor subtypes, AT1a and AT1b, and of AT2 receptors were detected. In conclusion, Ang II modulates the spontaneous contractility of rat jejunum via postjunctional AT1 receptors, involving Ca2+ mobilization from intracellular stores, via PLC/IP3 pathway, and Ca2+ influx from extracellular space, via L-type channels. Prejunctional AT2 receptors would counteract AT1 receptor effects, via NO synthesis. The observed age-related differences in the expression of all AT receptor subtypes are not reflected in the muscular contractile response to Ang II

Chemical Characterization and Cytotoxic and Antioxidant Activity Evaluation of the Ethanol Extract from the Bulbs of Pancratium maritimun Collected in Sicily

P. maritimum L., belonging to the Amaryllidaceae family, is a species that grows on beaches and coastal sand dunes mainly on both sides of the Mediterranean Sea and Black Sea, the Middle East, and up to the Caucasus region. It has been largely investigated due to its several interesting biological properties. With the aim of providing new insights into the phytochemistry and pharmacology of this species, the ethanolic extract of the bulbs from a local accession, not previously studied, growing in Sicily (Italy), was investigated. This chemical analysis, performed by mono- and bi-dimensional NMR spectroscopy, as well as LC-DAD-MSn, allowed to identify several alkaloids, three of which were never detected in the genus Pancratium. Furthermore, the cytotoxicity of the preparation was assessed in differentiated human Caco-2 intestinal cells by trypan blue exclusion assay, and its antioxidant potential was evaluated using the DCFH-DA radical scavenging method. The results obtained demonstrate that P. maritimum bulbs’ extract exerts no cytotoxic effect and is able to remove free radicals at all the concentrations tested

Uncovering the signaling pathway behind extracellular guanine-induced activation of NO System: New perspectives in memory-related disorders

Mounting evidence suggests that the guanine-based purines stand out as key player in cell metabolism and in several models of neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases. Guanosine (GUO) and guanine (GUA) are extracellular signaling molecules derived from the breakdown of the correspondent nucleotide, GTP, and their intracellular and extracellular levels are regulated by the fine-tuned activity of two major enzymes, purine nucleoside phosphorylase (PNP) and guanine deaminase (GDA). Noteworthy, GUO and GUA, seem to play opposite roles in the modulation of cognitive functions, such as learning and memory. Indeed GUO, despite exerting neuroprotective, anti-apoptotic and neurotrophic effects, causes a decay of cognitive activities, whereas GUA administration in rats results in working memory improvement (prevented by L-NAME pre-treatment). This study was designed to investigate, in a model of SH-SY5Y neuroblastoma cell line, the signal transduction pathway activated by extracellular GUA. Altogether, our results showed that: (i) in addition to an enhanced phosphorylation of ASK1, p38 and JNK, likely linked to a non-massive and transient ROS production, the PKB/NO/sGC/cGMP/PKG/ERK cascade seems to be the main signaling pathway elicited by extracellular GUA; (ii) the activation of this pathway occurs in a pertussis-toxin sensitive manner, thus suggesting the involvement of a putative G protein coupled receptor; (iii) the GUA-induced NO production, strongly reduced by cell pre-treatment with L-NAME, is negatively modulated by the EPAC-cAMP-CaMKII pathway, which causes the over-expression of GDA that, in turn, reduces the levels of GUA. These molecular mechanisms activated by GUA may be useful to support our previous observation showing that GUA improves learning and memory functions through the stimulation of NO signaling pathway, and underscore the therapeutic potential of oral administration of guanine for treating memory-related disorders

Indicaxanthin Induces Autophagy in Intestinal Epithelial Cancer Cells by Epigenetic Mechanisms Involving DNA Methylation

Autophagy is an evolutionarily conserved process critical in maintaining cellular homeostasis. Recently, the anticancer potential of autophagy inducers, including phytochemicals, was suggested. Indicaxanthin is a betalain pigment found in prickly pear fruit with antiproliferative and pro-apoptotic activities in colorectal cancer cells associated with epigenetic changes in selected methylation-silenced oncosuppressor genes. Here, we demonstrate that indicaxanthin induces the up-regulation of the autophagic markers LC3-II and Beclin1, and increases autophagolysosome production in Caco-2 cells. Methylomic studies showed that the indicaxanthin-induced pro-autophagic activity was associated with epigenetic changes. In addition to acting as a hypermethylating agent at the genomic level, indicaxanthin also induced significant differential methylation in 39 out of 47 autophagy-related genes, particularly those involved in the late stages of autophagy. Furthermore, in silico molecular modelling studies suggested a direct interaction of indicaxanthin with Bcl-2, which, in turn, influenced the function of Beclin1, a key autophagy regulator. External effectors, including food components, may modulate the epigenetic signature of cancer cells. This study demonstrates, for the first time, the pro-autophagic potential of indicaxanthin in human colorectal cancer cells associated with epigenetic changes and contributes to outlining its potential healthy effect in the pathophysiology of the gastrointestinal tract

Control of enteric neuromuscular functions by purinergic P2X7 receptors in normal rat distal colon and experimental bowel inflammation

Introduction: Purinergic signalling plays a pivotal role in the physiological regulation of several enteric functions, as well as in the modulation of immune/inflammatory cell activity. Recent evidence has shown an active involvement of the purinergic P2X7 receptor (P2X7R) in the fine tuning of immune functions, as well as its critical role in driving enteric neuron apoptosis under intestinal inflammation. However, the participation of this receptor pathway in the regulation of enteric neuromuscular functions remains undetermined. Aims: This study investigated the role of P2X7Rs in the control of colonic motility, both under normal conditions and in the presence of experimental colitis. Methods: Colitis was induced by intrarectal administration of 2,4-dinitrobenzenesulfonic acid (DNBS) in adult male Sprague-Dawley rats. Six days after colitis induction, colonic longitudinal muscle strips (LMS), obtained from normal or inflamed rats, were suspended in organ baths, containing Krebs solution additioned with NK 1, 2 and 3 receptor antagonists, and connected to isometric transducers to record atropine-sensitive cholinergic motor activity. The effects of A804598 (selective P2X7R antagonist; 0.001-100 μM) and BzATP (selective P2X7R agonist; 0.001-10 μM) were tested on contractions evoked by electrical stimulation (ES: 0.5 ms, 28 V, 10 Hz), delivered as single train (sES) or repeated every 60 s (rES), or by carbachol (1 μM) in the presence of tetrodotoxin. Results: In normal LMS, A804598 induced a negligible enhancing effect on sES-induced contractions (+7.8±3.5% at 0.1 μM), while a significant increase in the contractile responses elicited by sES was recorded in LMS from inflamed animals (+42.5±3.9% at 0.1 μM). Incubation of LMS with the adrenergic blocker guanethidine (10 μM) did not affect the enhancing effect exerted by A804598 in the presence of colitis. Upon incubation with Nw-propyl-L-arginine (NPA, inhibitor of neuronal nitric oxide synthase), which per se increased the sES-induced contractions in both normal and inflamed LMS preparations (+15.1±5.5% in normal and +143.5±9.5% in inflamed animals), the A804598 effects were lost. The pharmacological activation of P2X7Rs with BzATP did not significantly affect the contractions to rES in normal LMS (Emax= -10.2±1.9%), while a marked reduction was recorded in LMS from animals with colitis (Emax= -30.5±2.2%). The inhibitory effect of BzATP was antagonized by A804598, and it was also markedly blunted by NPA. Both P2X7R ligands did not affect carbachol-induced contractions. Conclusions: The purinergic system contributes to functional neuromuscular changes associated with bowel inflammation through the involvement of P2X7Rs, which modulate the activity of excitatory cholinergic nerves via a facilitatory control on inhibitory nitrergic pathways