IFN-α/ß-mediated NK2R expression is related to the malignancy of colon cancer cells.

Neurokinin 2 receptor (NK2R), a G protein-coupled receptor for neurokinin A (NKA), a tachykinin family member, regulates various physiological functions including pain response, relaxation of smooth muscle, dilation of blood vessels, and vascular permeability. However, the precise role and regulation of NK2R expression in cancer cells have not been fully elucidated. In this study, we found that high NK2R gene expression was correlated with the poor survival of colorectal cancer patients, and Interferon (IFN-α/ß) stimulation significantly enhanced NK2R gene expression level of colon cancer cells in a Janus kinas 1/2 (JAK 1/2)-dependent manner. NKA stimulation augmented viability/proliferation and phosphorylation of Extracellular-signal-regulated kinase 1/2 (ERK1/2) levels of IFN-α/ß-treated colon cancer cells and NK2R blockade by using a selective antagonist reduced the proliferation in vitro. Administration of an NK2R antagonist alone or combined with polyinosinic-polycytidylic acid, a synthetic analog of double-stranded RNA, to CT26-bearing mice significantly suppressed tumorigenesis. NK2R-overexpressing CT26 cells showed enhanced tumorigenesis and metastatic colonization in both lung and liver after the inoculation into mice. These findings indicate that IFN-α/ß-mediated NK2R expression is related to the malignancy of colon cancer cells, suggesting that NK2R blockade may be a promising strategy for colon cancers.

Congenital ablation of Tacr2 reveals overlapping and redundant roles of NK2R signaling in the control of reproductive axis.

Tachykinin (TAC) signaling is an important element in the central control of reproduction. TAC family is mainly composed of substance P (SP), neurokinin A (NKA), and NKB, which bind preferentially to NK1, NK2, and NK3 receptors, respectively. While most studies have focused on the reproductive functions of NKB/NK3R, and to a lesser extent SP/NK1R, the relevance of NK2R, encoded by Tacr2, remains poorly characterized. Here, we address the physiological roles of NK2R in regulating the reproductive axis by characterizing a novel mouse line with congenital ablation of Tacr2. Activation of NK2R evoked acute luteinizing hormone (LH) responses in control mice, similar to those of agonists of NK1R and NK3R. Despite the absence of NK2R, Tacr2-/- mice displayed only partially reduced LH responses to an NK2R agonist, which, nonetheless, were abrogated after blockade of NK3R in Tacr2-/- males. While Tacr2-/- mice displayed normal pubertal timing, LH pulsatility was partially altered in Tacr2-/- females in adulthood, with suppression of basal LH levels, but no changes in the number of LH pulses. In addition, trends for increase in breeding intervals were detected in Tacr2-/- mice. However, null animals of both sexes were fertile, with no changes in estrous cyclicity or sex preference in social behavioral tests. In conclusion, stimulation of NK2R elicited LH responses in mice, while congenital ablation of Tacr2 partially suppressed basal and stimulated LH secretion, with moderate reproductive impact. Our data support a modest, albeit detectable, role of NK2R in the control of the gonadotropic axis, with partially overlapping and redundant functions with other tachykinin receptors.NEW & NOTEWORTHY We have explored here the impact of congenital ablation of the gene (Tacr2) encoding the tachykinin receptor, NK2R, in terms of neuroendocrine control of the reproductive axis, using a novel Tacr2 KO mouse line. Our data support a modest, albeit detectable, role of NK2R in the control of the gonadotropic axis, with partially overlapping and redundant functions with other tachykinin receptors.

Gender-Related Differences of Tachykinin NK2 Receptor Expression and Activity in Human Colonic Smooth Muscle.

The tachykinin NK2 receptor plays a key role in gastrointestinal motor function. Enteric neurons release neurokinin A (NKA), which activates NK2 receptors on gastrointestinal smooth muscle, leading to contraction and increased motility. In patients with diarrhea-predominant irritable bowel syndrome, the NK2 receptor antagonist ibodutant had a greater therapeutic effect in females than males. The present study aimed to determine whether gender influences the expression and activity of NK2 receptors in human colonic smooth muscle. In vitro functional studies were performed to examine the contractile responses of colonic muscle strips to NKA and the selective NK2 receptor agonist [Lys5,MeLeu9,Nle10]NKA(4-10). Contractions were also measured in the presence of ibodutant to determine its antagonistic potency. The signal transduction pathways coupled to NK2 receptor activation were investigated using second messenger inhibitors. Western blot and fluorescent immunohistochemistry were conducted to determine the protein expression and localization of NK2 receptors. NK2 receptor-mediated contractility was greater in females compared with males. When against NKA, ibodutant was more potent in females. NK2 receptor expression increased with age in females, but not in males. Phospholipase C-mediated signaling was less prominent in females compared with males, whereas Ca2+ sensitization via Rho kinase and protein kinase C appeared to be the dominant pathway in both genders. The distribution of NK2 receptors in the human colon did not differ between the genders. Overall, gender differences exist in the expression and activity of NK2 receptors in colonic smooth muscle. These gender distinctions should be considered in the therapeutic development of NK2 receptor agents. SIGNIFICANCE STATEMENT: The tachykinin NK2 receptor has been identified as a therapeutic target for the treatment of bowel and bladder dysfunctions. The present study has revealed gender-related variations in NK2 receptor activity, signaling transduction pathways, antagonist potency, and changes in expression with age. These factors may underlie the gender differences in the treatment of diarrhea-predominant irritable bowel syndrome with NK2 receptor antagonists. Our findings highlight that gender differences should be considered in the therapeutic development of NK2 receptor agents.

The role of neurokinin A and its receptor in the regulation of prolactin secretion by the anterior pituitary of cyclic pigs.

In pigs, plasma prolactin concentration markedly changes during the oestrous cycle and the regulation of its secretion is very complex. The contribution of neurokinins in this process has not been sufficiently delineated. The aim of the study was to examine the effects of neurokinin A (NKA) on prolactin synthesis and secretion in cyclic gilts. The expression of NKA precursor (Ppta) and receptor (Tacr2) genes as well as NKA and TACR proteins content in the porcine pituitaries (days 2-3, 9-10, 12-13, 15-16 and 19-20 of the cycle) was determined. Furthermore, the in vitro influence of NKA on the expression of prolactin (Prl), dopamine receptor (D2r), TRH receptor (Trhr) genes and prolactin secretion by the porcine pituitary cells (days 9-10, 15-16 and 19-20 of the cycle) was assessed. The expression of Ppta and Tacr2 as well as NKA and TACR proteins in the pituitary tissue has been changing throughout the oestrous cycle. NKA affected in vitro the expression of studied genes and prolactin secretion depending on the stage of the cycle, dose of NKA and/or duration of the cell incubation. Altogether, the study indicates that NKA is engaged in the modulation of prolactin secretion in the pig during the oestrous cycle.

Characterization of the Role of NKA in the Control of Puberty Onset and Gonadotropin Release in the Female Mouse.

The tachykinin neurokinin B (NKB, Tac2) is critical for proper GnRH release in mammals, however, the role of the other tachykinins, such as substance P (SP) and neurokinin A (NKA) in reproduction, is still not well understood. In this study, we demonstrate that NKA controls the timing of puberty onset (similar to NKB and SP) and stimulates LH release in adulthood through NKB-independent (but kisspeptin-dependent) mechanisms in the presence of sex steroids. Furthermore, this is achieved, at least in part, through the autosynaptic activation of Tac1 neurons, which express NK2R (Tacr2), the receptor for NKA. Conversely, in the absence of sex steroids, as observed in ovariectomy, NKA inhibits LH through a mechanism that requires the presence of functional receptors for NKB and dynorphin (NK3R and KOR, respectively). Moreover, the ability of NKA to modulate LH secretion is absent in Kiss1KO mice, suggesting that its action occurs upstream of Kiss1 neurons. Overall, we demonstrate that NKA signaling is a critical component in the central control of reproduction, by contributing to the indirect regulation of kisspeptin release.

DNA methylation of the Tacr2 gene in a CUMS model of depression.

Tacr2, the gene encoding the NK2 receptor, belongs to G protein-coupled receptors. Accumulating evidence has indicated that the tachykinin receptors may contribute to the pathophysiology of depression. During the last decade, some studies have shown that Tacr2 activation is involved in the modulation of emotional processes. However, the extent, to which stress impacts Tacr2 expression remains unclear. The molecular mechanisms underlying depression also remain poorly understood. In this study, we subjected adult male Sprague Dawley (SD) rats to chronic unpredictable mild stress (CUMS) to induce a depression-like phenotype. We then measured the body weight and performed the sucrose preference test, forced swimming test (FST) and open field test to detect the effects of stress on anhedonia and activity. Western blotting and real-time PCR were used to study the protein and mRNA expression levels of Tacr2, respectively, in the hypothalamus. To explore DNA methylation of the Tacr2 gene, we used methylated DNA immunoprecipitation sequencing (MeDIP-seq). Additionally, we used the bisulfite sequencing PCR (BSP) to further verify the DNA methylation levels of the Tacr2 receptor gene in rats. We found that the CUMS-sensitive rats exhibited a decrease in body weight and sucrose preference, a decrease in the distance traveled, rearing frequency and velocity in the open field test, and an increase in immobility time in the FST. Compared with the expression in the control rats, Tacr2 protein and mRNA expression in the hypothalamus significantly increased in the CUMS-sensitive rats; however, the DNA methylation levels of the Tacr2 gene were significantly lower than in the control rats. In summary, according to our findings, the stress-induced increase in Tacr2 expression in the hypothalamus correlated with a specific decrease in DNA methylation of the Tacr2 gene. These results may enrich the understanding of the pathological processes of depression and provide insights into therapeutic approaches for its treatment.

Gastric ulcer induced changes in substance P and Nk1, Nk2, Nk3 receptors expression in different stomach localizations with regard to intrinsic neuronal system.

Gastric ulceration, a focal tissue damage accompanied by inflammation, can influence other parts of the stomach. Substance P and its receptors are strongly involved in regulation of gastrointestinal motility, secretion and inflammation. The enteric nervous system is one of the regulators of gastrointestinal functioning and contributes to tissue response to the pathology. The pig, an omnivorous animal, is a valuable species for gastrointestinal experiments. Thus, the objective of the study was to verify whether the antral ulceration induces changes in the expression of substance P and tachykinin receptors in the neighboring (antrum) and distanced (corpus, pylorus) porcine gastric tissues and therein localized myenteric and submucosal perikarya as well as in the intrinsic descending neurons supplying pyloric sphincter. The experiment was performed on healthy pigs and pigs with experimentally induced gastric ulcers. Stomach samples from the corpus, antrum (adjacent to the ulcer in experimental pigs) and pylorus were analyzed by: (1) double immunofluorescence for changes in the number of SP-positive myenteric and submucosal neurons (2) Real-Time PCR for changes in expression of mRNA encoding SP and Nk1, Nk2, Nk3 receptors. Additionally, gastric descending neurons supplying pyloric sphincter were immunostained for SP. In experimental animals, only the number of SP-positive myenteric perikarya significantly increased in all stomach localizations studied. Q-PCR revealed increased expression for: SP, Nk1, Nk3 in the corpus; Nk2 and Nk3 in the pylorus; In the antrum, expression of Nk3 was increased but Nk2-decreased. Antral ulcers induced significant changes in the expression of SP and tachykinin receptors in the wide stomach area indicating sophisticated tissue reaction.

Improved ligand-binding- and signaling-competent human NK2R yields in yeast using a chimera with the rat NK2R C-terminus enable NK2R-G protein signaling platform.

The tachykinin 2 receptor (NK2R) plays critical roles in gastrointestinal, respiratory and mental disorders and is a well-recognized target for therapeutic intervention. To date, therapeutics targeting NK2R have failed to meet regulatory agency approval due in large part to the limited characterization of the receptor-ligand interaction and downstream signaling. Herein, we report a protein engineering strategy to improve ligand-binding- and signaling-competent human NK2R that enables a yeast-based NK2R signaling platform by creating chimeras utilizing sequences from rat NK2R. We demonstrate that NK2R chimeras incorporating the rat NK2R C-terminus exhibited improved ligand-binding yields and downstream signaling in engineered yeast strains and mammalian cells, where observed yields were better than 4-fold over wild type. This work builds on our previous studies that suggest exchanging the C-termini of related and well-expressed family members may be a general protein engineering strategy to overcome limitations to ligand-binding and signaling-competent G protein-coupled receptor yields in yeast. We expect these efforts to result in NK2R drug candidates with better characterized signaling properties.

Communication Between Enteric Neurons, Glia, and Nociceptors Underlies the Effects of Tachykinins on Neuroinflammation.

Background & Aims: Tachykinins are involved in physiological and pathophysiological mechanisms in the gastrointestinal tract. The major sources of tachykinins in the gut are intrinsic enteric neurons in the enteric nervous system and extrinsic nerve fibers from the dorsal root and vagal ganglia. Although tachykinins are important mediators in the enteric nervous system, how they contribute to neuroinflammation through effects on neurons and glia is not fully understood. Here, we tested the hypothesis that tachykinins contribute to enteric neuroinflammation through mechanisms that involve intercellular neuron-glia signaling. Methods: We used immunohistochemistry and quantitative real-time polymerase chain reaction, and studied cellular activity using transient-receptor potential vanilloid-1 (TRPV1)tm1(cre)Bbm/J::Polr2atm1(CAG-GCaMP5g,-tdTomato)Tvrd and Sox10CreERT2::Polr2atm1(CAG-GCaMP5g,-tdTomato)Tvrd mice or Fluo-4. We used the 2,4-di-nitrobenzene sulfonic acid (DNBS) model of colitis to study neuroinflammation, glial reactivity, and neurogenic contractility. We used Sox10::CreERT2+/-/Rpl22tm1.1Psam/J mice to selectively study glial transcriptional changes. Results: Tachykinins are expressed predominantly by intrinsic neuronal varicosities whereas neurokinin-2 receptors (NK2Rs) are expressed predominantly by enteric neurons and TRPV1-positive neuronal varicosities. Stimulation of NK2Rs drives responses in neuronal varicosities that are propagated to enteric glia and neurons. Antagonizing NK2R signaling enhanced recovery from colitis and prevented the development of reactive gliosis, neuroinflammation, and enhanced neuronal contractions. Inflammation drove changes in enteric glial gene expression and function, and antagonizing NK2R signaling mitigated these changes. Neurokinin A-induced neurodegeneration requires glial connexin-43 hemichannel activity. Conclusions: Our results show that tachykinins drive enteric neuroinflammation through a multicellular cascade involving enteric neurons, TRPV1-positive neuronal varicosities, and enteric glia. Therapies targeting components of this pathway could broadly benefit the treatment of dysmotility and pain after acute inflammation in the intestine.

Ablation of Tacr2 in mice leads to gastric emptying disturbance.

BACKGROUND: Tacr2 is one of the G protein-coupled receptors(GPCRs) that mediate the biological actions of tachykinins. It is abundantly expressed in the gastrointestinal (GI) system and is thought to play an important role in GI motility, secretion, and visceral sensitivity. Previously, the physiological and pathophysiological functions of Tacr2 were mainly studied using Tacr2 selective agonists or antagonists. Here, we seek to investigate the effect of Tacr2 disruption in mice to provide further insights. METHODS: The Tacr2 knockout mice were generated by homologous recombination and the phenotypic changes of the Tacr2-null mice were analyzed and compared with their wild type (wt) littermates. KEY RESULTS: Increased food retention was detected in Tacr2-/- mice. The stomach of Tacr2-/- mice had thinner muscularis externa and less neurons in the myenteric plexus. The stomach and small intestine exhibited longer duration of electrical field stimulation (EFS)-induced inhibition in the gastric fundus and decreased frequency of migrating motor complex (MMC), respectively. Neuronal nitric oxide synthase (nNOS) and vasoactive intestinal polypeptide (VIP) were significantly up-regulated due to Tarc2 deficiency, contributing to enhanced nitric oxide (NO) signaling in the stomach of Tacr2-/- mice. Intraperitoneal application of 7-nitroindazole (7-NI) to Tacr2-/- mice effectively relieved the gastric emptying disturbance. Moreover, Creb and NF-κB signalings were involved in the regulation of these physiological changes initiated by Tacr2 deficiency. CONCLUSIONS & INFERENCES: Tacr2 negatively regulated the expression of nNOS and VIP both in vivo and in vitro. Its ablation in mice elevated the expression of nNOS and VIP, enhanced NO signaling and changed the Creb and NF-κB signalings, finally leading to the gastric emptying disturbance of Tacr2-/- mice.

Intratracheal Administration of Mesenchymal Stem Cells Modulates Tachykinin System, Suppresses Airway Remodeling and Reduces Airway Hyperresponsiveness in an Animal Model.

BACKGROUND: The need for new options for chronic lung diseases promotes the research on stem cells for lung repair. Bone marrow-derived mesenchymal stem cells (MSCs) can modulate lung inflammation, but the data on cellular processes involved in early airway remodeling and the potential involvement of neuropeptides are scarce. OBJECTIVES: To elucidate the mechanisms by which local administration of MSCs interferes with pathophysiological features of airway hyperresponsiveness in an animal model. METHODS: GFP-tagged mouse MSCs were intratracheally delivered in the ovalbumin mouse model with subsequent functional tests, the analysis of cytokine levels, neuropeptide expression and histological evaluation of MSCs fate and airway pathology. Additionally, MSCs were exposed to pro-inflammatory factors in vitro. RESULTS: Functional improvement was observed after MSC administration. Although MSCs did not adopt lung cell phenotypes, cell therapy positively affected airway remodeling reducing the hyperplastic phase of the gain in bronchial smooth muscle mass, decreasing the proliferation of epithelium in which mucus metaplasia was also lowered. Decrease of interleukin-4, interleukin-5, interleukin-13 and increase of interleukin-10 in bronchoalveolar lavage was also observed. Exposed to pro-inflammatory cytokines, MSCs upregulated indoleamine 2,3-dioxygenase. Moreover, asthma-related in vivo upregulation of pro-inflammatory neurokinin 1 and neurokinin 2 receptors was counteracted by MSCs that also determined a partial restoration of VIP, a neuropeptide with anti-inflammatory properties. CONCLUSION: Intratracheally administered MSCs positively modulate airway remodeling, reduce inflammation and improve function, demonstrating their ability to promote tissue homeostasis in the course of experimental allergic asthma. Because of a limited tissue retention, the functional impact of MSCs may be attributed to their immunomodulatory response combined with the interference of neuropeptide system activation and tissue remodeling.

Altered expression of the tachykinins substance P/neurokinin A/hemokinin-1 and their preferred neurokinin 1/neurokinin 2 receptors in uterine leiomyomata.

OBJECTIVE: To study the expression levels of tachykinins and tachykinin receptors in uterine leiomyomas and matched myometrium. DESIGN: Laboratory study. SETTING: University research laboratories and academic hospital. PATIENT(S): Women undergoing hysterectomy for symptomatic leiomyomas. INTERVENTION(S): Quantitative polymerase chain reaction, immunohistochemistry and Western blot. MAIN OUTCOME MEASURE(S): Expression and tissue immunostaining of substance P, neurokinin A, hemokinin-1, neurokinin 1 receptor full-length (NK1R-Fl) and truncated (NK1R-Tr) isoforms, and neurokinin 2 receptor (NK2R) in paired samples of leiomyoma and adjacent normal myometrium. RESULT(S): TAC1 messenger RNA (mRNA) was significantly up-regulated in leiomyomas, whereas intense immunoreaction for the three peptides was particularly abundant in connective tissue cells. Differential regulation of TACR1 mRNA was observed, and at the protein level there was a significant increased expression of NK1R short isoform (NK1R-Tr). TACR2 mRNA was significantly up-regulated in leiomyomas, although levels of NK2R protein were similar in normal and tumor cells. CONCLUSION(S): These and our previous data demonstrate that the whole tachykinin system is differentially regulated in leiomyomas. The increased expression of NK1R-Tr might stimulate leiomyoma growth in a similar way to that observed in other steroid-dependent tumors.

Prenatal nicotinic exposure upregulates pulmonary C-fiber NK1R expression to prolong pulmonary C-fiber-mediated apneic response.

Prenatal nicotinic exposure (PNE) prolongs bronchopulmonary C-fiber (PCF)-mediated apneic response to intra-atrial bolus injection of capsaicin in rat pups. The relevant mechanisms remain unclear. Pulmonary substance P and adenosine and their receptors (neurokinin-A receptor, NK1R and ADA1 receptor, ADA1R) and transient receptor potential cation channel subfamily V member 1 (TRPV1) expressed on PCFs are critical for PCF sensitization and/or activation. Here, we compared substance P and adenosine in BALF and NK1R, ADA1R, and TRPV1 expression in the nodose/jugular (N/J) ganglia (vagal pulmonary C-neurons retrogradely labeled) between Ctrl and PNE pups. We found that PNE failed to change BALF substance P and adenosine content, but significantly upregulated both mRNA and protein TRPV1 and NK1R in the N/J ganglia and only NK1R mRNA in pulmonary C-neurons. To define the role of NK1R in the PNE-induced PCF sensitization, the apneic response to capsaicin (i.v.) without or with pretreatment of SR140333 (a peripheral and selective NK1R antagonist) was compared and the prolonged apnea by PNE significantly shortened by SR140333. To clarify if the PNE-evoked responses depended on action of nicotinic acetylcholine receptors (nAChRs), particularly α7nAChR, mecamylamine or methyllycaconitine (a general nAChR or a selective α7nAChR antagonist) was administrated via another mini-pump over the PNE period. Mecamylamine or methyllycaconitine eliminated the PNE-evoked mRNA and protein responses. Our data suggest that PNE is able to elevate PCF NK1R expression via activation of nAChRs, especially α7nAChR, which likely contributes to sensitize PCFs and prolong the PCF-mediated apneic response to capsaicin.

Substance P Receptor Antagonist Suppresses Inflammatory Cytokine Expression in Human Disc Cells.

STUDY DESIGN: Laboratory study. OBJECTIVE: To evaluate whether blockade of the Substance P (SP) NK1R attenuates its proinflammatory effect on human intervertebral disc cells (IVD), and to evaluate the signaling pathways associated with SP. SUMMARY OF BACKGROUND DATA: SP and its receptors are expressed in human IVD cells, and cause upregulation of inflammatory mediators; however, the effects of blocking these receptors have not been studied in human IVD cells. METHODS: Human annulus fibrosus (AF) and nucleus pulposus (NP) cells were expanded in monolayer, and then suspended in alginate beads. The alginate beads were treated with culture medium first containing a high affinity NK1R antagonist (L-760735) at different concentrations, and then with medium containing both NK1R antagonist and SP at 2 concentrations. Ribonucleic acid was isolated and transcribed into cDNA. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to evaluate expression of interleukin (IL)-1ß, IL-6, and IL-8. Western blot analysis was performed to examine levels of the phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal regulated kinase 1/2 (ERK1/2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB p65). The cells were pretreated with specific inhibitors of p38 (SB203580), ERK1/2 (PD98059), and p65 (SM7368) and then stimulated with SP. RESULTS: We detected expression of NK1R, neurokinin receptor 2 (NK2R), and neurokinin receptor 3 (NK3R) in AF and NP cells. Treatment of disc cells with the NK1R antagonist was able to suppress expression of IL-1ß, IL-6, and IL-8 in a dose-dependent manner. SP stimulation increased phosphorylation of p38-MAPK and ERK1/2, but not of NFκB p65. This indicates that p38-MAPK and ERK1/2 control SP-induced cytokine expression independently from NF-kB p65. Inhibition of p38 and ERK1/2 activation reduced SP-induced IL-6 production in human disc cells. CONCLUSION: NK1R is responsible for the proinflammatory effect of SP on IVD cells and this effect can be blocked by preventing binding of SP to NK1R. This study shows for the first time that SP mediates signaling in disc cells through NK1R and that SP activates the proinflammatory p38-MAPK and ERK1/2 pathways. LEVEL OF EVIDENCE: 4.

A Fluorescent Live Imaging Screening Assay Based on Translocation Criteria Identifies Novel Cytoplasmic Proteins Implicated in G Protein-coupled Receptor Signaling Pathways.

Several cytoplasmic proteins that are involved in G protein-coupled receptor signaling cascades are known to translocate to the plasma membrane upon receptor activation, such as beta-arrestin2. Based on this example and in order to identify new cytoplasmic proteins implicated in the ON-and-OFF cycle of G protein-coupled receptor, a live-imaging screen of fluorescently labeled cytoplasmic proteins was performed using translocation criteria. The screening of 193 fluorescently tagged human proteins identified eight proteins that responded to activation of the tachykinin NK2 receptor by a change in their intracellular localization. Previously we have presented the functional characterization of one of these proteins, REDD1, that translocates to the plasma membrane. Here we report the results of the entire screening. The process of cell activation was recorded on videos at different time points and all the videos can be visualized on a dedicated website. The proteins BAIAP3 and BIN1, partially translocated to the plasma membrane upon activation of NK2 receptors. Proteins ARHGAP12 and PKM2 translocated toward membrane blebs. Three proteins that associate with the cytoskeleton were of particular interest : PLEKHH2 rearranged from individual dots located near the cell-substrate adhesion surface into lines of dots. The speriolin-like protein, SPATC1L, redistributed to cell-cell junctions. The Chloride intracellular Channel protein, CLIC2, translocated from actin-enriched plasma membrane bundles to cell-cell junctions upon activation of NK2 receptors. CLIC2, and one of its close paralogs, CLIC4, were further shown to respond with the same translocation pattern to muscarinic M3 and lysophosphatidic LPA receptors. This screen allowed us to identify potential actors in signaling pathways downstream of G protein-coupled receptors and could be scaled-up for high-content screening.

Changes in gene expression and methylation in the blood of patients with first-episode psychosis.

Schizophrenia is a severe mental health disorder with high heritability. The investigation of individuals during their first-episode psychosis (FEP), before the progression of psychotic disorders and especially before treatment with antipsychotic medications, is particularly helpful for understanding this complex disease and for the identification of potential biomarkers. In this study, we compared the expression of genes that are involved in neurotransmission and neurodevelopment of antipsychotic-naive FEP in the peripheral blood of patients (n=51) and healthy controls (n=51). In addition, we investigated the differentially expressed genes with respect to a) DNA methylation, b) the correlation between gene expression and clinical variables (PANSS), and c) gene expression changes after risperidone treatment. Expression levels of 11 genes were quantified with SYBR Green. For methylation analysis, bisulfite sequencing was performed. A significant decrease in GCH1 mRNA levels was observed in FEP patients relative to controls. Also, when we compare the FEP patients after risperidone treatment with controls, this difference remains significant, and no significant differences were observed in GCH1 mRNA levels when comparing patients before and after risperidone treatment. Additionally, although the differences were non-significant after Bonferroni correction, the expression of GCH1 seemed to be correlated with PANSS scores, and the GCH1 promoter region was more methylated in FEP than in controls, thus corroborating the results obtained at the mRNA level. Few studies have been conducted on GCH1, and future studies are needed to clarify its potential role in the progression of schizophrenia.

Novel pituitary actions of TAC3 gene products in fish model: receptor specificity and signal transduction for prolactin and somatolactin α regulation by neurokinin B (NKB) and NKB-related peptide in carp pituitary cells.

TAC3 is a member of tachykinins, and its gene product neurokinin B (NKB) has recently emerged as a key regulator for LH through modulation of kisspeptin/GnRH system within the hypothalamus. In fish models, TAC3 not only encodes NKB but also a novel tachykinin-like peptide called NKB-related peptide (NKBRP), and the pituitary actions of these TAC3 gene products are still unknown. Using grass carp as a model, the direct effects and postreceptor signaling for the 2 TAC3 products were examined at the pituitary level. Grass carp TAC3 was cloned and confirmed to encode NKB and NKBRP similar to that of other fish species. In carp pituitary cells, NKB and NKBRP treatment did not affect LH release and gene expression but up-regulated prolactin (PRL) and somatolactin (SL)α secretion, protein production, and transcript expression. The stimulation by these 2 TAC3 gene products on PRL and SLα release and mRNA levels were mediated by pituitary NK2 and NK3 receptors, respectively. Apparently, NKB- and NKBRP-induced SLα secretion and transcript expression were caused by adenylate cyclase/cAMP/protein kinase A, phospholipase C/inositol 1,4,5-triphosphate/protein kinase C and Ca(2+)/calmodulin/Ca(2+)/calmodulin-dependent protein kinase II activation. The signal transduction for the corresponding responses on PRL release and mRNA expression were also similar, except that the protein kinase C component was not involved. These findings suggest that the 2 TAC3 gene products do not play a role in LH regulation at the pituitary level in carp species but may serve as novel stimulators for PRL and SLα synthesis and secretion via overlapping postreceptor signaling mechanisms coupled to NK2 and NK3 receptors, respectively.

Are biological actions of neurokinin A in the adult brain mediated by a cross-talk between the NK1 and NK2 receptors?

Mice lacking the NK(1) receptor (NK(1)R-/- mice) and selective, high-affinity, non-peptide, NK(1), NK(2) and NK(3) receptor antagonists were used to identify the tachykinin receptor subtype(s) mediating the central responses induced by neurokinin A (NKA). The peptides, substance P (SP), NKA and senktide and the antagonists were injected intracerebroventricularly (ICV) through an implanted cannula. NKA (50 pmol) was as potent as SP (50 pmol) in inducing grooming behaviour (face washing and hind limb grooming) in wild-type mice, but both peptides failed to induce behavioural responses in NK(1)R-/- mice. In wild-type mice, the NK(1) receptor antagonist, RP 67580 (2 nmol), effectively inhibited grooming behaviour elicited by SP, but was inactive against grooming induced by NKA, which in turn was abolished after pre-treatment with the selective NK(2) receptor agonist, SR 48968 (2 nmol). Unlike NKA, the selective NK(2) receptor agonists, (ß Ala(8)) NKA 4-10 and (NLeu(10)) NKA 4-10, injected ICV at doses of 50 or 100 pmol did not elicit any behavioural response in wild-type mice. The NK(3) receptor antagonist, SR 142801, inhibited behaviours induced by the NK(3) receptor agonist, senktide, but did not alter behavioural responses to either SP or NKA in wild-type mice. The present findings demonstrate that central biological actions of SP and senktide are mediated by activation of NK(1) and NK(3) receptors, respectively. Our results also indicate that NK(1) receptors are essential for generating central actions induced by NKA, which are most probably mediated by a cross-talk between the NK(1) and NK(2) receptors.

Neuropeptide signaling activates dendritic cell-mediated type 1 immune responses through neurokinin-2 receptor.

Neurokinin A (NKA), a neurotransmitter distributed in the central and peripheral nervous system, strictly controls vital responses, such as airway contraction, by intracellular signaling through neurokinin-2 receptor (NK2R). However, the function of NKA-NK2R signaling on involvement in immune responses is less-well defined. We demonstrate that NK2R-mediated neuropeptide signaling activates dendritic cell (DC)-mediated type 1 immune responses. IFN-γ stimulation significantly induced NK2R mRNA and remarkably enhanced surface protein expression levels of bone marrow-derived DCs. In addition, the DC-mediated NKA production level was significantly elevated after IFN-γ stimulation in vivo and in vitro. We found that NKA treatment induced type 1 IFN mRNA expressions in DCs. Transduction of NK2R into DCs augmented the expression level of surface MHC class II and promoted Ag-specific IL-2 production by CD4(+) T cells after NKA stimulation. Furthermore, blockade of NK2R by an antagonist significantly suppressed IFN-γ production by both CD4(+) T and CD8(+) T cells stimulated with the Ag-loaded DCs. Finally, we confirmed that stimulation with IFN-γ or TLR3 ligand (polyinosinic-polycytidylic acid) significantly induced both NK2R mRNA and surface protein expression of human PBMC-derived DCs, as well as enhanced human TAC1 mRNA, which encodes NKA and Substance P. Thus, these findings indicate that NK2R-dependent neuropeptide signaling regulates Ag-specific T cell responses via activation of DC function, suggesting that the NKA-NK2R cascade would be a promising target in chronic inflammation caused by excessive type 1-dominant immunity.

Tachykinins stimulate a subset of mouse taste cells.

The tachykinins substance P (SP) and neurokinin A (NKA) are present in nociceptive sensory fibers expressing transient receptor potential cation channel, subfamily V, member 1 (TRPV1). These fibers are found extensively in and around the taste buds of several species. Tachykinins are released from nociceptive fibers by irritants such as capsaicin, the active compound found in chili peppers commonly associated with the sensation of spiciness. Using real-time Ca(2+)-imaging on isolated taste cells, it was observed that SP induces Ca(2+) -responses in a subset of taste cells at concentrations in the low nanomolar range. These responses were reversibly inhibited by blocking the SP receptor NK-1R. NKA also induced Ca(2+)-responses in a subset of taste cells, but only at concentrations in the high nanomolar range. These responses were only partially inhibited by blocking the NKA receptor NK-2R, and were also inhibited by blocking NK-1R indicating that NKA is only active in taste cells at concentrations that activate both receptors. In addition, it was determined that tachykinin signaling in taste cells requires Ca(2+)-release from endoplasmic reticulum stores. RT-PCR analysis further confirmed that mouse taste buds express NK-1R and NK-2R. Using Ca(2+)-imaging and single cell RT-PCR, it was determined that the majority of tachykinin-responsive taste cells were Type I (Glial-like) and umami-responsive Type II (Receptor) cells. Importantly, stimulating NK-1R had an additive effect on Ca(2+) responses evoked by umami stimuli in Type II (Receptor) cells. This data indicates that tachykinin release from nociceptive sensory fibers in and around taste buds may enhance umami and other taste modalities, providing a possible mechanism for the increased palatability of spicy foods.