Industrial-produced lemon nanovesicles ameliorate experimental colitis-associated damages in rats via the activation of anti-inflammatory and antioxidant responses and microbiota modification.

Plant-derived nanovesicles (PDNVs) have recently emerged as natural delivery systems of biofunctional compounds toward mammalian cells. Considering their already described composition, anti-inflammatory properties, stability, and low toxicity, PDNVs offer a promising path for developing new preventive strategies for several inflammatory diseases, among which the inflammatory bowel disease (IBD). In this study, we explore the protective effects of industrially produced lemon vesicles (iLNVs) in a rat model of IBD. Characterization of iLNVs reveals the presence of small particles less than 200 nm in size and a profile of bioactive compounds enriched in flavonoids and organic acids with known beneficial properties. In vitro studies on human macrophages confirm the safety and anti-inflammatory effects of iLNVs, as evidenced by the reduced expression of pro-inflammatory cytokines and increased levels of anti-inflammatory markers. As evidenced by in vivo experiments, pre-treatment with iLNVs significantly alleviates symptoms and histological features in 2,4 dinitrobenzensulfuric acid (DNBS)-induced colitis in rats. Molecular pathway analysis reveals modulation of NF-κB and Nrf2, indicating anti-inflammatory and antioxidant effects. Finally, iLNVs affects gut microbiota composition, improving the consistent colitis-related alterations. Overall, we demonstrated the protective role of industrially produced lemon nanovesicles against colitis and emphasized their potential in managing IBD through multifaceted mechanisms.

Motor dysfunction of the gut in Duchenne muscular dystrophy: A review.

BACKGROUND: Duchenne's muscular dystrophy (DMD) is a severe type of hereditary, neuromuscular disorder caused by a mutation in the dystrophin gene resulting in the absence or production of truncated dystrophin protein. Conventionally, clinical descriptions of the disorder focus principally on striated muscle defects; however, DMD manifestations involving gastrointestinal (GI) smooth muscle have been reported, even if not rigorously studied. PURPOSE: The objective of the present review is to offer a comprehensive perspective on the existing knowledge concerning GI manifestations in DMD, focusing the attention on evidence in DMD patients and mdx mice. This includes an assessment of symptomatology, etiological pathways, and potential corrective approaches. This paper could provide helpful information about DMD gastrointestinal implications that could serve as a valuable orientation for prospective research endeavors in this field. This manuscript emphasizes the effectiveness of mdx mice, a DMD animal model, in unraveling mechanistic insights and exploring the pathological alterations in the GI tract. The gastrointestinal consequences evident in patients with DMD and the mdx mice models are a significant area of focus for researchers. The exploration of this area in depth could facilitate the development of more efficient therapeutic approaches and improve the well-being of individuals impacted by the condition.

Inhibitory effect and underlying mechanism of essential oil of Prangos ferulacea Lindl (L.) on spontaneous and induced uterine contractions in non-pregnant rats.

Evidence suggests the use of natural compounds as support in the management of uterine contractility disorders. We recently demonstrated that the essential oil of Apiacea Prangos ferulacea (L.) (Prangoil) modulates intestinal smooth muscle contractility. Thus, we aimed to evaluate if Prangoil could also affect the contractility of uterine muscle in non-pregnant rat and to investigate the related action mechanism/s. The effects of the aromatic monoterpenes, ß-ocimene and carvacrol, constituents of Prangoil, were also evaluated. Spontaneous contractions and contraction-induced by K+-depolarization and oxytocin in rat uterus were recorded in vitro, using organ bath technique. Prangoil reduced the amplitude of spontaneous contractions as well as responses to KCl and oxytocin. ß-ocimene and carvacrol matched oil inhibitory effects. Prangoil effects were not affected by nitrergic and adenylyl cyclase inhibitors or non-specific potassium channel blocker, but they were reduced by nifedipine, L-type calcium channel inhibitor, or 2-aminoethoxydiphenylborate (2-APB), membrane-permeant inositol 1,4,5-triphosphate receptor inhibitor. The response to ß-ocimene was reduced by nifedipine and by 2-APB (20 µM), whilst carvacrol inhibitory effect was attenuated only by nifedipine. In conclusion, Prangoil, and its components, ß-ocimene and carvacrol, reduced spontaneous and KCl or oxytocin-induced contractions of rat myometrium, mainly modulating extracellular Ca2+ influx through L-Type channels and Ca2+ release from the intracellular store. Further studies could contribute to evaluate the potential use of Prangoil against disorders characterized by abnormal uterine contractions.

Essential oil of Sicilian Prangos ferulacea (L.) Lindl. and its major component, ß-ocimen, affect contractility in rat small and large intestine.

ETHNOPHARMACOLOGICAL RELEVANCE: Prangos ferulacea (L.) Lindl is an Apiaceae plant, widely used in traditional medicine. Recently, chemical composition and biological activities of its essential oil (Prangroil) have been reported, but there are no studies on possible effects on intestinal contractility. AIMS OF THE STUDY: We investigated the effects of essential oil Sicilian Prangoil on the contractility of rat small (duodenum) and large (colon) intestine and the related action mechanism. MATERIALS AND METHODS: Responses to Prangoil and to its major component ß-ocimen in intestinal segments were assessed in vitro as changes in isometric tension. RESULTS: Prangoil, induced in duodenum, depending upon doses, contraction and/or muscular relaxation. Instead, in colon Prangoil only reduced the phasic contractions and induced muscular relaxation. ß-ocimen, in both segments, produced only reduction of the spontaneous contractions without affecting basal tone. Prangoil contractile effects were abolished by ω-conotoxin, neural N-type Ca2+ channels blocker, atropine, muscarinic receptor antagonist, neostigmine, acetylcholinesterase (AChE) inhibitor, suggesting that Prangoil-induced contraction would be the result of an increase in neuronal cholinergic activity. Prangoil and ß-ocimen inhibitory effects were unaffected by ω-conotoxin, L-NAME, blocker of the NO synthase, ODQ, soluble guanylate cyclase inhibitor, excluding involvement of neurotransmitter release or NO synthesis in the inhibitory effects. Potassium channel blocker did not affect Prangoil or ß-ocimen inhibitory responses. Prangoil or ß-ocimen inhibited the Ca2+ and high-KCl solution -induced contractions and the Carbachol-induced contractions in calcium free solution. CONCLUSION: Prangoil affects the contractility of small and large intestine in rat, with regional differences, via potentiation of neural cholinergic activity, blockade of L-type voltage-gated calcium channel and reduction of Ca2+ release from the intracellular store. The Prangroil main components, ß-ocimen, contributes to the inhibitory effects.

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.

Inhibition of uterine contractility by guanine-based purines in non-pregnant rats.

Growing evidence pointed out that guanine-based purines are able to modulate smooth muscle contractile activity of blood vessels and gastrointestinal tract. Since, so far, possible guanine-based purine modulation of uterine musculature is unknown, the aim of the present study was to investigate in vitro, using organ bath technique, guanosine and guanine effects on spontaneous uterine contraction, and uterine contraction induced by K+-depolarization and oxytocin in a non-pregnant rat. Guanosine, but not guanine, reduced the amplitude of spontaneous contraction of the uterine muscle in a dose-dependent manner. The inhibitory response was antagonized by S-(4-nitrobenzyl)-6-thioinosine (NBTI), a membrane nucleoside transporter inhibitor, but persisted in the presence of theophylline, a nonselective adenosine receptor antagonist, or propanolol, ß1/ß2 adrenoreceptor antagonist or blockers of a nitrergic pathway. In addition, potassium channel blockers did not influence guanosine-induced effects. Guanosine was able to inhibit the external calcium (Ca2+) influx-induced contraction, but it did not affect the contraction induced by high-KCl solution, indicating that guanosine does not interact with L-type voltage-gated calcium channel. Guanosine prevented/reduced uterine contractions induced by oxytocin, even in the absence of external calcium. In conclusion, guanosine is able to reduce both spontaneous and oxytocin-induced contractions of rat myometrium, likely subsequently to its intracellular intake. The blockade of extracellular Ca2+ influx and reduction of Ca2+ release from the intracellular store are the mechanisms involved in the guanosine-induced tocolytic effects.

The multiple roles of dopamine receptor activation in the modulation of gastrointestinal motility and mucosal function.

Dopamine (DA) is a catecholamine regulatory molecule with potential role in physiology and physiopathology of the intestinal tract. Various cellular sources of DA have been indicated as enteric neurons, immune cells, intestinal flora and gastrointestinal epithelium. Moreover, DA is produced by nutritional tyrosine. All the five DA receptors, actually described, are present throughout the gut. Current knowledge of DA in this area is reviewed, focusing on gastrointestinal function in health and during inflammation. Research on animal models and humans are reported. A major obstacle to understanding the physiologic and/or pharmacological roles of enteric DA is represented by the multiplicity of receptors involved in the responses together with many signalling pathways related to each receptor subtype. It is mandatory to map precisely the distributions of DA receptors, to determine the relevance of a receptor in a specific location in order to explore novel therapies directed to dopaminergic targets that may be useful in the control of intestinal inflammation.

Role of cyclooxygenase pathways in bowel fibrotic remodelling in a murine model of experimental colitis.

OBJECTIVE: Gut fibrosis occurs under chronic inflammation. This study examined the effects of different cyclooxygenase (COX) inhibitors on fibrosis in the inflamed colon. METHODS: Colitis was induced by 2,4-dinitrobenzenesulfonic acid (DNBS) in albino male Sprague-Dawley rats. After 6, 12 and 18 days, macroscopic and microscopic damage, collagen and elastic fibre content were examined. At day 6, pro-fibrotic factors (collagen I and III, hydroxyproline, fibronectin, matrix metalloproteinase-2 and -9), transforming growth factor-beta (TGF-ß) signalling [TGF-ß, Ras homolog gene family member A (RhoA), phosphorylated small mother against decapentaplegic (pSMAD)-2 and -6] and peristalsis were assessed, and the effects of indomethacin, SC-560 or celecoxib were tested. KEY FINDINGS: Six days after DNBS administration, significant histopathological signs of fibrotic remodelling were observed in rats. At day 6, pro-fibrotic factors were up-regulated and colonic peristalsis was altered. COX inhibitors reversed the histochemical, molecular and functional changes in the fibrotic colon. COX inhibition reduced TGF-ß expression, SMAD2 phosphorylation and RhoA, and increased SMAD6 expression. CONCLUSIONS: Colonic fibrosis is associated with altered bowel motility and induction of profibrotic factors driven by TGF-ß signalling. COX-1 and COX-2 inhibition counteracts this fibrotic remodelling by the modulation of TGF-ß/SMAD signalling, mainly via SMAD6 induction and reduction in SMAD2 phosphorylation.

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. (AU)

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.

Anti-Inflammatory Potential of Brassicaceae-Derived Phytochemicals: In Vitro and In Vivo Evidence for a Putative Role in the Prevention and Treatment of IBD.

Inflammatory bowel disease (IBD) is a group of intestinal disorders, of unknown etiology, characterized by chronic inflammation within the gut. They are gradually becoming critical because of the increasing incidence worldwide and improved diagnosis. Due to the important side effects observed during conventional therapy, natural bioactive components are now under intense investigation for the prevention and treatment of chronic illnesses. The Brassicaceae family comprises vegetables widely consumed all over the world. In recent decades, a growing body of literature has reported that extracts from the Brassicaceae family and their purified constituents have anti-inflammatory properties, which has generated interest from both the scientific community and clinicians. In this review, data from the literature are scrutinized and concisely presented demonstrating that Brassicaceae may have anti-IBD potential. The excellent biological activities of Brassicacea are widely attributable to their ability to regulate the levels of inflammatory and oxidant mediators, as well as their capacity for immunomodulatory regulation, maintenance of intestinal barrier integrity and intestinal flora balance. Possible future applications of bioactive-derived compounds from Brassicaceae for promoting intestinal health should be investigated.

Age-related differences of γ-aminobutyric acid (GABA)ergic transmission in human colonic smooth muscle.

BACKGROUND: Enteric neurons undergo to functional changes during aging. We investigated the possible age-associated differences in enteric γ-aminobutyric acid (GABA)ergic transmission evaluating function and distribution of GABAergic receptors in human colon. METHODS: Mechanical responses to GABA and GABA receptor agonists on slow phasic contractions were examined in vitro as changes in isometric tension in colonic muscle strips from young (<65 years old) and aged patients (>65 years old). GABAergic receptor expression was assessed by quantitative RT-PCR. KEY RESULTS: In both preparations GABA induced an excitatory effect, consisting in an increase in the basal tone, antagonized by the GABAA receptor antagonist, bicuculline, and potentiated by phaclofen, GABAB receptor antagonist.Tetrodotoxin (TTX) and atropine-sensitive contractile responses to GABA and GABAA receptor agonist, muscimol, were more pronounced in old compared to young subjects. Baclofen, GABAB receptor agonist, induced a TTX-sensitive reduction of the amplitude of the spontaneous. Nω-nitro-l-arginine methyl ester (L-NAME), nitric oxide (NO) synthase inhibitor abolished the inhibitory responses in old preparations, but a residual responses persisted in young preparations, which in turn was abolished by suramin, purinergic receptor antagonist. α3-GABAA receptor subunit expression tends to change in an age-dependent manner. CONCLUSIONS AND INFERENCES: Our results reveal age-related differences in GABAergic transmission in human colon. At all the age tested GABA regulates muscular contractility modulating the activity of the intrinsic neurons. Activation of GABAA receptor, through acetylcholine release, induces contraction, which increases in amplitude with age. GABAB receptor activation leads to neural release of NO and purines, being a loss of purinergic-component in aged group.

AphaMax®, an Aphanizomenon Flos-Aquae Aqueous Extract, Exerts Intestinal Protective Effects in Experimental Colitis in Rats.

BACKGROUND: Aphanizomenon flos-aquae (AFA) is a unicellular cyanobacterium considered to be a "superfood" for its complete nutritional profile and beneficial properties. We investigated possible beneficial effects of an AFA extract, commercialized as AphaMax®, containing concentrated amount of phycocyanins and phytochrome, in 2,4 dinitrobenzensulfonic acid(DNBS)-induced colitis in rats. METHODS: Effects of preventive oral treatment of AphaMax® (20, 50 or 100 mg/kg/day) in colitic rats were assessed and then macroscopic and microscopic analyses were performed to evaluate the inflammation degree. Myeloperoxidase (MPO) activity and NF-κB, pro-inflammatory citockines, cycloxygenase-2 (COX-2), and inducible NOS (iNOS) levels of expression were determined, as Reactive Oxygen Species (ROS) and nitrite levels. RESULTS: AphaMax® treatment attenuated the severity of colitis ameliorating clinical signs. AphaMax® reduced the histological colonic damage and decreased MPO activity, NF-κB activation, as well as iNOS and COX-2 expression. AphaMax® treatment improved the altered immune response associated with colonic inflammation reducing IL-1ß, IL-6 expression. Lastly, AphaMax® reduced oxidative stress, decreasing ROS and nitrite levels. CONCLUSIONS: Preventive treatment with AphaMax® attenuates the severity of the inflammation in DNBS colitis rats involving decrease of the NF-kB activation, reduction of iNOS and COX-2 expression, and inhibition of oxidative stress. Due its anti-inflammatory and antioxidant proprieties AphaMax® could be a good candidate as a complementary drug in inflammatory bowel disease (IBD) treatment.

Opposite effects of dopamine on the mechanical activity of circular and longitudinal muscle of human colon.

BACKGROUND: Because dopamine (DA) has gained increasing evidence as modulator of gut motility, we aimed to characterize dopaminergic response in human colon, evaluating function and distribution of dopamine receptors in circular vs longitudinal muscle strips. METHODS: Mechanical responses to DA and dopaminergic agonists on slow phasic contractions and on basal tone were examined in vitro as changes in isometric tension. RT-PCR was used to reveal the distribution of dopaminergic receptors. KEY RESULTS: In spontaneous active circular muscle, DA induced an increase in the amplitude of slow phasic contractions and of the basal tone, via activation of D1-like receptors. DA contractile responses were insensitive to neural blockers or to atropine and inhibited by phospholipase C (PLC) pathway inhibitors. In precontracted circular muscle strips, DA, at the higher concentrations tested, caused a relaxant response via activation of D2-like receptors. In the longitudinal muscle, DA caused only muscular relaxation due to activation of D2-like receptors. DA relaxant responses were insensitive to neural blockers or to nitric oxide synthase inhibitor and reduced by a wide-spectrum K+ channel blockers. Transcripts encoding for all the dopaminergic receptor subtypes was observed in both circular and longitudinal preparations. CONCLUSIONS AND INFERENCES: Dopamine is able to modulate contractile activity of the human colon. In the circular muscle layer, DA induces mainly muscular contraction activating non-neural D1-like receptors, coupled to PLC/IP3 pathway. In the longitudinal muscle layer, DA induces muscular relaxation acting on non-neural D2-like receptors leading to the increase in K+ conductance.

PD123319, angiotensin II type II receptor antagonist, inhibits oxidative stress and inflammation in 2, 4-dinitrobenzene sulfonic acid-induced colitis in rat and ameliorates colonic contractility.

Angiotensin II, the main effector of renin angiotensin system, plays an important role in the inflammatory process and most of its effects are mediated through the AT1 receptor activation. However, the knowledge about the AT2 receptor involvement in this process is still evolving. We previously found that in an experimental model of colitis, AT2 receptor activation can contribute to the impairment of the muscle contractility in vitro in the course of inflammation. Here, we investigated the potential alleviating effects of the in vivo treatment of PD123319 (1-[[4-(Dimethylamino)-3-methylphenyl]methyl]-5-(diphenylacetyl)-4,5,6,7- tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid ditrifluoroacetate), AT2 receptor antagonist, in 2,4-dinitrobenzene sulfonic acid (DNBS)-induced rat model of colitis. The effects of i.p PD123319 (0.3, 3 and 10 mg/kg) administration to rats subjected to intra-rectal DNBS instillation were investigated. The study revealed that the colon injury and the inflammatory signs were ameliorated by PD123319 when visualized by the histopathological examination. The colon shortening, myeloperoxidase activity, and colonic expression of IL-1ß, IL-6 and iNOS were downregulated in a dose-dependent manner in DNBS-induced colitis rats treated with PD123319 and the anti-oxidant defense machinery was also improved. The mechanism of these beneficial effects was found in the ability of PD123319 to inhibit NF-κB activation induced by DNBS. The colonic contractility in inflamed tissues was also improved by PD123319 treatment. In conclusion, our data have demonstrated previously that undescribed proinflammatory effects for the AT2 receptors in DNBS-induced colitis in rats in which they are mediated likely by NF-κB activation and reactive oxygen species generation. Moreover, when the inflammatory process is mitigated by the AT2 receptor antagonist treatment, the smooth muscle is able to recover its functionality.

Preventive effects of guanosine on intestinal inflammation in 2, 4-dinitrobenzene sulfonic acid (DNBS)-induced colitis in rats.

BACKGROUND: Guanosine, a guanine-based purine, is an extracellular signaling molecule exerting anti-inflammatory and antioxidative effects in several in vivo and in vitro injury models. We aimed to investigate its protective effects on 2, 4-dinitrobenzene sulfonic acid (DNBS)-induced colitis in rat. METHODS: Rats were divided into five groups and colitis was induced by intracolonic instillation of DNBS (15 mg/rat). Guanosine (4 or 8 mg/kg) was administered for 6 days i.p. starting the day of the colitis induction. Body weight loss, stool consistency, colon weight/length, histological analysis, myeloperoxidase activity (MPO) and pro-inflammatory cytokine levels were assessed. Immunoblotting of nuclear factor-κB (NF-κB) p65 protein levels and detection of oxidative and nitrosative stress markers were also performed. RESULTS: Guanosine, in a dose-dependent manner, significantly ameliorated the severity of DNBS-induced colitis, reducing body weight loss and diarrhea incidence, preventing the DNBS-induced macroscopic and microscopic damage to the colonic mucosa, and the MPO increase. Guanosine treatment also lowered interleukin-1ß, interleukin-6, and tumor necrosis factor-α mRNA levels. Importantly, guanosine in DNBS rats down-regulated the expression of NF-κB p65 and the levels of reactive oxygen species and nitrite. CONCLUSIONS: In conclusion, guanosine exerts beneficial effects in DNBS-induced colitis in rats, through modulation of colonic inflammation, downregulating of NFκB-mediated signaling.

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.

Altered gastrointestinal motility in an animal model of Lesch-Nyhan disease.

Mutations in the HGPRT1 gene, which encodes hypoxanthine-guanine phosphoribosyltransferase (HGprt), housekeeping enzyme responsible for recycling purines, lead to Lesch-Nyhan disease (LND). Clinical expression of LND indicates that HGprt deficiency has adverse effects on gastrointestinal motility. Therefore, we aimed to evaluate intestinal motility in HGprt knockout mice (HGprt¯). Spontaneous and neurally evoked mechanical activity was recorded in vitro as changes in isometric tension in circular muscle strips of distal colon. HGprt¯ tissues showed a lower in amplitude spontaneous activity and atropine-sensitivity neural contraction compared to control mice. The responses to carbachol and to high KCl were reduced, demonstrating a widespread impairment of contractility. L-NAME was not able in the HGprt¯ tissues to restore the large amplitude contractile activity typical of control. In HGprt¯ colon, a reduced expression of dopaminergic D1 receptor was observed together with the loss of its tonic inhibitory activity present in control-mice. The analysis of inflammatory and oxidative stress in colonic tissue of HGprt¯ mice revealed a significant increase of lipid peroxidation associated with over production of oxygen free radicals. In conclusion, HGprt deficiency in mice is associated with a decrease in colon contractility, not dependent upon reduction of acetylcholine release from the myenteric plexus or hyperactivity of inhibitory signalling. By contrast the increased levels of oxidative stress could partially explain the reduced colon motility in HGprt¯ mice. Colonic dysmotility observed in HGprt¯ mice may mimic the gastrointestinal dysfunctions symptoms of human syndrome, providing a useful animal model to elucidate the pathophysiology of this problem in the LND.

Dopamine induces inhibitory effects on the circular muscle contractility of mouse distal colon via D1- and D2-like receptors

Dopamine (DA) acts as gut motility modulator, via D1- and D2-like receptors, but its effective role is far from being clear. Since alterations of the dopaminergic system could lead to gastrointestinal dysfunctions, a characterization of the enteric dopaminergic system is mandatory. In this study, we investigated the role of DA and D1- and D2-like receptors in the contractility of the circular muscle of mouse distal colon by organ-bath technique. DA caused relaxation in carbachol-precontracted circular muscle strips, sensitive to domperidone, D2-like receptor antagonist, and mimicked by bromocriptine, D2-like receptor agonist. 7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH-23390), D1-like receptor antagonist, neural toxins, L-NAME (nitric oxide (NO) synthase inhibitor), 2'-deoxy-N6-methyl adenosine 3',5'-diphosphate diammonium salt (MRS 2179), purinergic P2Y1 antagonist, or adrenergic antagonists were ineffective. DA also reduced the amplitude of neurally evoked cholinergic contractions. The effect was mimicked by (±)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrobromide (SKF-38393), D1-like receptor agonist and antagonized by SCH-23390, MRS 2179, or L-NAME. Western blotting analysis determined the expression of DA receptor proteins in mouse distal colon. Notably, SCH-23390 per se induced an increase in amplitude of spontaneous and neurally evoked cholinergic contractions, unaffected by neural blockers, L-NAME, MRS 2179, muscarinic, adrenergic, or D2-like receptor antagonists. Indeed, SCH-23390-induced effects were antagonized by an adenylyl cyclase blocker. In conclusion, DA inhibits colonic motility in mice via D2- and D1-like receptors, the latter reducing acetylcholine release from enteric neurons, involving nitrergic and purinergic systems. Whether constitutively active D1-like receptors, linked to adenylyl cyclase pathway, are involved in a tonic inhibitory control of colonic contractility is questioned