Differential roles of serotonin receptor subtypes in regulation of neurotrophin receptor expression and intestinal hypernociception.

OBJECTIVES: Aberrant serotonin (5-hydroxytryptamine, 5-HT) metabolism and neurite outgrowth were associated with abdominal pain in irritable bowel syndrome (IBS). We previously demonstrated that 5-HT receptor subtype 7 (5-HT7) was involved in visceral hypersensitivity of IBS-like mouse models. The aim was to compare the analgesic effects of a novel 5-HT7 antagonist to reference standards in mouse models and investigate the mechanisms of 5-HT7-dependent neuroplasticity. METHODS: Two mouse models, including Giardia post-infection combined with water avoidance stress (GW) and post-resolution of trinitrobenzene sulfonic acid-induced colitis (PT) were used. Mice were orally administered CYY1005 (CYY, a novel 5-HT7 antagonist), alosetron (ALN, a 5-HT3 antagonist), and loperamide (LPM, an opioid receptor agonist) prior to measurement of visceromotor responses (VMR). Levels of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin receptors (NTRs) were assessed. RESULTS: Peroral CYY was more potent than ALN or LPM in reducing VMR values in GW and PT mice. Increased mucosal 5-HT7-expressing nerve fibers were associated with elevated Gap43 levels in the mouse colon. We observed higher colonic Ntrk2 and Ngfr expression in GW mice, and increased Bdnf expression in PT mice compared with control mice. Human SH-SY5Y cells stimulated with mouse colonic supernatant or exogenous serotonin exhibited longer nerve fibers, which CYY dose-dependently inhibited. Serotonin increased Ntrk1 and Ngfr expression via 5-HT7 but not 5-HT3 or 5-HT4, while Ntrk2 upregulation was dependent on all three 5-HT receptor subtypes. CONCLUSIONS: Stronger analgesic effects by peroral CYY were observed compared with reference standards in two IBS-like mouse models. The 5-HT7-dependent NTR upregulation and neurite elongation may be involved in intestinal hypernociception.

5-HT7 receptor-dependent intestinal neurite outgrowth contributes to visceral hypersensitivity in irritable bowel syndrome.

Irritable bowel syndrome (IBS) is characterized by visceral hypersensitivity (VH) associated with abnormal serotonin/5-hydroxytryptamine (5-HT) metabolism and neurotrophin-dependent mucosal neurite outgrowth. The underlying mechanisms of VH remain poorly understood. We investigated the role of 5-HT7 receptor in mucosal innervation and intestinal hyperalgesia. A high density of mucosal nerve fibres stained for 5-HT7 was observed in colonoscopic biopsy specimens from IBS patients compared with those from healthy controls. Staining of 5-HT3 and 5-HT4 receptors was observed mainly in colonic epithelia with comparable levels between IBS and controls. Visceromotor responses to colorectal distension were evaluated in two mouse models, one postinfectious with Giardia and subjected to water avoidance stress (GW) and the other postinflammatory with trinitrobenzene sulfonic acid-induced colitis (PT). Increased VH was associated with higher mucosal density of 5-HT7-expressing nerve fibres and elevated neurotrophin and neurotrophin receptor levels in the GW and PT mice. The increased VH was inhibited by intraperitoneal injection of SB-269970 (a selective 5-HT7 antagonist). Peroral multiple doses of CYY1005 (a novel 5-HT7 ligand) decreased VH and reduced mucosal density of 5-HT7-expressing nerve fibres in mouse colon. Human neuroblastoma SH-SY5Y cells incubated with bacteria-free mouse colonic supernatant, 5-HT, nerve growth factor, or brain-derived neurotrophic factor exhibited nerve fibre elongation, which was inhibited by 5-HT7 antagonists. Gene silencing of HTR7 also reduced the nerve fibre length. Activation of 5-HT7 upregulated NGF and BDNF gene expression, while stimulation with neurotrophins increased the levels of tryptophan hydroxylase 2 and 5-HT7 in neurons. A positive-feedback loop was observed between serotonin and neurotrophin pathways via 5-HT7 activation to aggravate fibre elongation, whereby 5-HT3 and 5-HT4 had no roles. In conclusion, 5-HT7-dependent mucosal neurite outgrowth contributed to VH. A novel 5-HT7 antagonist could be used as peroral analgesics for IBS-related pain.

The Intraocular Pressure Lowering Effect of a Dual Kinase Inhibitor (ITRI-E-(S)4046) in Ocular Hypertensive Animal Models.

Purpose: The purpose of this study was to develop a preclinical compound, ITRI-E-(S)4046, a dual synergistic inhibitor of myosin light chain kinase 4 (MYLK4) and Rho-related protein kinase (ROCK), for reducing intraocular pressure (IOP). Methods: ITRI-E-(S)4046 is an amino-pyrazole derivative with physical and chemical properties suitable for ophthalmic formulation. In vitro kinase inhibition was evaluated using the Kinase-Glo Luminescent Kinase Assays. A comprehensive kinase selectivity analysis of ITRI-E-(S)4046 was performed using the KINOMEscan assay from DiscoverRx. The IOP reduction and tolerability of ITRI-E-(S)4046 were assessed in ocular normotensive rabbits, ocular normotensive non-human primates, and ocular hypertensive rabbits. In vivo studies were conducted to assess drug concentrations in ocular tissue. The adverse ocular effects of rabbit eyes were evaluated following the OECD405 guidelines. Results: ITRI-E-(S)4046 showed highly selective kinase inhibitory activity against ROCK1/2, MYLK4, and mitogen-activated protein kinase kinase kinase 19 (MAP3K19), with high specificity against protein kinase A, G, and C families. In ocular normotensive rabbits and non-human primates, the mean IOP reductions of 0.1% ITRI-E-(S)4046 eye drops were 29.8% and 28.5%, respectively. In hypertonic saline-induced and magnetic beads-induced ocular hypertensive rabbits, the mean IOP reductions of ITRI-E-(S)4046 0.1% eye drops were 46.9% and 22.0%, respectively. ITRI-E-(S)4046 was well tolerated with only temporary and minor signs of hyperemia. Conclusions: ITRI-E-(S)4046 is a novel type of highly specific ROCK1/2 and MYLK4 inhibitor that can reduce IOP in normotensive and hypertensive animal models. It has the potential to become an effective and well-tolerated treatment for glaucoma.

Gut-derived cholecystokinin contributes to visceral hypersensitivity via nerve growth factor-dependent neurite outgrowth.

BACKGROUND AND AIM: Irritable bowel syndrome is characterized by abdominal pain and altered bowel habits and may occur following stressful events or infectious gastroenteritis such as giardiasis. Recent findings revealed a link between cholecystokinin (CCK), neurotrophin synthesis, and intestinal hyperalgesia. The aim was to investigate the role of CCK in visceral hypersensitivity using mouse models challenged with a bout of infection with Giardia lamblia or psychological stress, either alone or in combination. METHODS: Abdominal pain was evaluated by visceromoter response to colorectal distension. Nerve fibers in intestinal tissues were stained using immunohistochemistry (PGP9.5). Human neuroblastoma SH-SY5Y cells incubated with bacterial-free mouse gut supernatant or recombinant CCK-8S were assessed for neurite outgrowth and nerve growth factor (NGF) production. RESULTS: Intestinal hypersensitivity was induced by either stress or Giardia infection, and a trend of increased pain was seen following dual stimuli. Increased CCK levels and PGP9.5 immunoreactivity were found in colonic mucosa of mice following stress and/or infection. Inhibitors to the CCK-A receptor (L-364718) or CCK-B receptor (L-365260) blocked visceral hypersensitivity caused by stress, but not when induced by giardiasis. Nerve fiber elongation and NGF synthesis were observed in SH-SY5Y cells after incubation with colonic supernatants from mice given the dual stimuli, or after treatment with CCK-8S. Increased nerve fiber length by colonic supernatant and CCK-8S was attenuated by L-365260 or neutralizing anti-NGF. CONCLUSIONS: This new model successfully recapitulates intestinal hypernociception induced by stress or Giardia. Colonic CCK contributes to visceral hypersensitivity caused by stress, but not by Giardia, partly via NGF-dependent neurite outgrowth.