Nitric Oxide: From Gastric Motility to Gastric Dysmotility

It is known that nitric oxide (NO) plays a key physiological role in the control of gastrointestinal (GI) motor phenomena. In this respect, NO is considered as the main non-adrenergic, non-cholinergic (NANC) inhibitory neurotransmitter responsible for smooth muscle relaxation. Moreover, many substances (including hormones) have been reported to modulate NO production leading to changes in motor responses, further underlying the importance of this molecule in the control of GI motility. An impaired NO production/release has indeed been reported to be implicated in some GI dysmotility. In this article we wanted to focus on the influence of NO on gastric motility by summarizing knowledge regarding its role in both physiological and pathological conditions. The main role of NO on regulating gastric smooth muscle motor responses, with particular reference to NO synthases expression and signaling pathways, is discussed. A deeper knowledge of nitrergic mechanisms is important for a better understanding of their involvement in gastric pathophysiological conditions of hypo- or hyper-motility states and for future therapeutic approaches. A possible role of substances which, by interfering with NO production, could prove useful in managing such motor disorders has been advanced

Platelet-Rich Plasma Modulates Gap Junction Functionality and Connexin 43 and 26 Expression During TGF-β1-Induced Fibroblast to Myofibroblast Transition: Clues for Counteracting Fibrosis

Skeletal muscle repair/regeneration may benefit by Platelet-Rich Plasma (PRP) treatment owing to PRP pro-myogenic and anti-fibrotic effects. However, PRP anti-fibrotic action remains controversial. Here, we extended our previous researches on the inhibitory effects of PRP on in vitro transforming growth factor (TGF)-β1-induced differentiation of fibroblasts into myofibroblasts, the effector cells of fibrosis, focusing on gap junction (GJ) intercellular communication. The myofibroblastic phenotype was evaluated by cell shape analysis, confocal fluorescence microscopy and Western blotting analyses of α-smooth muscle actin and type-1 collagen expression, and electrophysiological recordings of resting membrane potential, resistance, and capacitance. PRP negatively regulated myofibroblast differentiation by modifying all the assessed parameters. Notably, myofibroblast pairs showed an increase of voltage-dependent GJ functionality paralleled by connexin (Cx) 43 expression increase. TGF-β1-treated cells, when exposed to a GJ blocker, or silenced for Cx43 expression, failed to differentiate towards myofibroblasts. Although a minority, myofibroblast pairs also showed not-voltage-dependent GJ currents and coherently Cx26 expression. PRP abolished the TGF-β1-induced voltage-dependent GJ current appearance while preventing Cx43 increase and promoting Cx26 expression. This study adds insights into molecular and functional mechanisms regulating fibroblast-myofibroblast transition and supports the anti-fibrotic potential of PRP, demonstrating the ability of this product to hamper myofibroblast generation targeting GJs

Tumor necrosis factor αlpha impairs kisspeptin signaling in human fetal hypothalamic neurons

  Previous studies in both human and animal models have linked overnutrition-related metabolic dysfunctions to hypothalamic inflammation (1, 2). Moreover, metabolic disorders are often associated with male hypogonadotropic hypogonadism, suggesting that inflammatory pathways may impair central regulatory networks involving gonadotropin releasing hormone (GnRH) neuron activity. However, studies in humans are strongly hampered by the anatomical distribution of these neurons, scattered within the preoptic area of the hypothalamus. This study was aimed at investigating the effects of inflammatory stimuli in human fetal hypothalamic (hfHypo) primary cell cultures. These cells have shown evident GnRH neuron features, as demonstrated by quantitative gene expression profile, immunohistochemistry, flow cytometry and ELISA detection of GnRH peptide in the culture medium. Moreover, hfHypo cells express KISS1R and accordingly respond to kisspeptin, the main central regulator of GnRH neuron function. Exposing hfHypo cells to TNFα (10ng/ml, 5h) determined nuclear translocation of NFkB, as well as a significant increase of COX2 mRNA expression, thus demonstrating the induction of inflammatory signaling. Prolonged exposure (24 h) to TNFα strongly down-regulated KISS1R mRNA without changing GnRH expression. Moreover, we know that kisspeptin is able to depolarize GnRH neurons through the activation of a canonical transient receptor potential (TRPC)-like cationic channel. Electrophisiological studies demonstrated that kisspeptin (1 μM) induced a clear TRPC-mediated depolarizing response in hfHypo cells. Pretreating cells with TNFα (10 ng/ml, 24h) significantly inhibited kisspeptin-sensitive TRPC currents. Our results indicate that inflammatory pathways may impair GnRH neuron function by interfering with the ability of these neurons in responding to kisspeptin

Isolation and characterization of neurons with a gonadotropin-releasing hormone (GnRH) phenotype from human foetal hypothalamus

GnRH neurons are a peculiar hypothalamic subpopulation crucially involved in the control of the reproductive axis. As well established in several animal models, the kisspeptin (KISS1)/KISS1 receptor (KSS1R) system plays a master role in the control of GnRH neurons, however, investigations in humans are strongly hampered by the anatomical distribution of these neurons, scattered within the preoptic area of the hypothalamus. This study was aimed at establishing a human hypothalamic primary cell culture with GnRH neuron features. Brains were recovered from 11-12 week-old human fetuses, then hypothalamic tissue, lining the 3rd ventricle, was dissected and processed for cell culture isolation. The primary cultures obtained were first characterized using flow cytometry and showed a mixed composition with the majority of cells (92±8 %) positive for the neuronal marker MAP2 and a low percentage of cells positive for the glial marker GFAP (13.5±9 %). Interestingly, among the neuronal population about 80% were GnRH-positive cells (77.8±20 %). Gene expression profiling, immunofluorescence and western blot analyses confirmed that these cells expressed GnRH, as well as KISS1R. Hence, electrophisiological studies were performed to investigate if these cells responded to kisspeptin (Kp) stimulation. Using the voltageclamp technique, we found that Kp (100nM or 1μM) induced a clear depolarizing response. Moreover, depolarizing effects of Kp involved transient receptor potential channels (TRPC), as expected by KISS1R activation (1). This is the first human hypothalamic cellular model with a GnRH neuron phenotype, representing a new tool for the investigation of human GnRH neuron biology