Fig 1: Analysis of HTR4 gene structure and identification of a novel transcript variant in brain. 5′ RACE in cDNA derived from total brain tissue indicated expression of transcripts possessing exons 1-3, and also a novel transcript in which exons 1 and 2 are replaced by an alternate exon (a). The remaining sequence of the novel transcript aligns with the equivalent region of transcript b (b). The predicted protein structure of this novel variant has an elongated N-terminus and affects the position of an N-linked glycosylation site in this region (c).
Fig 2: Activation of 5-HT4R inhibits mechanosensory activity of bladder afferent nerves. (A) Schematic of drug administration and original recordings from an ex vivo pelvic nerve recording preparation showing bladder afferent firing and intravesical pressure. Each vertical line represents a distension and the original record of the black vertical lines presented in the below. (B) Relationship between bladder afferent firing and intravesical pressure showing a significant decrease in afferent firing in the presence of RS67333 (10 μm) and reversal of the effect by GR125487. The averaged area under the curve (AUC) of Control (1,368 ± 80.8), RS67333 (762.3 ± 130.8), R&G (1,376 ± 77.38) were compared using one-way ANOVA. n = 6–8 mice for each group. (**, p = 0.002, RS67333 vs. Control.) (C) Effects of different concentrations of RS67333 (1, 3, 10 and 30 μm) on the mechanosensitivity of bladder afferent nerves. AUC analysis using one-way ANOVA: Control 1,319 ± 90.14; 1 μM 925.5 ± 53.10 (p = 0.0244 vs. control); 3 μm 768 ± 79.09 (p = 0.0029 vs. control); 10 μm 607 ± 112.7 (p = 0.0012 vs. control); 30 μm 498.1 ± 143.9 (p = 0.0003 vs. control). n = 4 mice for each group. (D) Effect of RS67333 and R&G (10 μm) on basal afferent firing (ns = not significant, one-way ANOVA, p > 0.05). (E) The relationship between intravesical pressure and volume (the compliance) during ramp bladder distension was unaffected by intravesical administration of RS67333 and R&G (10 μm, ns = not significant, one-way ANOVA, p > 0.05).
Fig 3: Confocal photomicrographs illustrating the colocalization of 5-HT4 receptor and CD31 proteins in the human hippocampus. 5-HT4 receptor (in green, Alexa 488, ⇨) and CD31 (in red, Alexa 568, ⇨) colocalize on the endothelial cells of capillary vessels and platelets (⇨). White full arrow indicates the nucleus of an endothelial cell; yellow arrow indicates a pericyte. The slight staining, in green and red, in the middle of the vessels, is due to the autofluorescence of hemoglobin.
Fig 4: The expression levels of 5-HT4R and the correlation between 5-HT4R and HERV-W env in recent-onset schizophrenia. (A) and (B) The concentration of 5-HT4R in healthy controls (N = 31) and schizophrenia (N = 33) by ELISA. (C) and (D) The concentration of HERV-W env in healthy controls (N = 31) and schizophrenia (N = 33) by ELISA. E Linear regression correlation between HERV-W env and 5-HT4R expression levels in schizophrenia. X-axis: the concentration of HERV-W env; Y-axis: the concentration of 5-HT4R. The line represents the calculated “best-fit” equation of values within the boxed area, with correlation value indicated on the top (R2 = 0.76). P < 0.01, P < 0.001 by median and nonparametric analysis. Note: each point generally represents a single patient, but a few are overlapping and cannot be separated on the graph.
Fig 5: Western blot analysis showing the involvement of the 5-HT4 receptor in the regulation of the expression of tight junction proteins in hCMEC/D3 cells. Cells were treated with GR113808 (1 µM), prucalopride (10 µM) or GR113808 (1 µM) + prucalopride (10 µM) for 96 h. Relative integrated density values of (a) ZO-1, (b) claudin-5 and (c) occludin are shown. No correlation is found between the treatment and the expression of ZO-1 and claudin-5. A significant negative correlation between treatment with prucalopride and the expression of occludin is apparent, compared to control. Histograms represent mean ± SEM from three independent experiments. * p < 0.05.
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