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Perineural invasion (PNI) is considered to be a main reason for the poor prognosis of pancreatic cancer. In the present study, we analyzed the roles of substance P (SP)/neurokinin-1 receptor (NK-1R) and lncRNA LOC389641 in pancreatic cancer PNI. Pancreatic cancer cell lines BxPC-3 and MIAPaCa-2 were cocultured with SH-SY5Y cells and then stimulated with SP to simulate the in vivo influence of ganglia on pancreatic cancer. The BxPC-3 and MIAPaCa-2 cells were transfected with a neurokinin-1 receptor (NK-1R) overexpression vector, NK-1R silencing vector, LOC389641 overexpression vector, or LOC389641 silencing vector, respectively. The proliferative abilities of BxPC-3 and MIAPaCa-2 cells were assessed using the cell counting kit-8 and 5-ethynyl-2′-deoxyuridine (EdU) assays. Wound-healing and Transwell assays were performed to determine the migration and invasion abilities of the cells. When SP was added to the coculture system, it positively regulated cancer cell proliferation, migration, and PNI and significantly activated the NK-1R/Akt/NF-κB signaling pathway. Incubation with 100 nmol/L SP for 24 h was selected as the optimal condition for treatment. The activated NK-1R positively regulated the proliferation, migration, and invasion of pancreatic cancer cells. However, the levels of lncRNA LOC389641 and tumor necrosis factor receptor SF10A (TNFRSF10A) mRNA in BxPC-3 and MIAPaCa-2 cells were not affected by SP treatment. Overexpression or silencing of LOC389641 changed the effect of SP stimulation on pancreatic cancer PNI. When taken together, these results revealed that SP/NK-1R and LOC389641 promoted the progression of pancreatic cancer PNI. Moreover, we found that pancreatic cancer PNI promoted by the SP/NK-1R axis could be blocked by the TNFRSF10A/NF-κB pathway mediated by LOC389641.

Background. Osteosarcoma, the most frequent osteogenic malignancy, has become a serious public health challenge due to its high morbidity rates and metastatic potential. Recently, the neurokinin-1 receptor (NK-1R) is proved to be a promising target in cancer therapy. This study is aimed at determining the effect of aprepitant, a safe and Food and Drug Administration (FDA) approved NK-1R antagonist, on osteosarcoma cell migration and metastasis, and to explore its underlying mechanism of action. Methods. Colorimetric MTT assay was employed to assess cell viability and cytotoxicity. A wound-healing assay was used to examine migration ability. The desired genes’ protein and mRNA expression levels were measured by western blot assay and quantitative real-time PCR (qRT-PCR), respectively. Gelatinase activity was also measured by zymography. Results. We found that aprepitant inhibited MG-63 osteosarcoma cell viability in a dose-dependent manner. We also observed that aprepitant inhibited the migrative phenotype of osteosarcoma cells and reduced the expression levels and activities of matrix metalloproteinases (MMP-2 and MMP-9). Aprepitant also reduced the expression of an angiogenic factor, VEGF protein, and NF-κB as an important transcriptional regulator of metastasis-related genes. Conclusion. Collectively, our observations indicate that aprepitant modulates the metastatic behavior of human osteosarcoma cells, which may be applied to an effective therapeutic approach for patients with metastatic osteosarcoma.

Protein-lipid interactions in cellular membranes modulate central cellular functions, are often transient in character, but occur too intermittently to be readily observable. We introduce transient state imaging (TRAST), combining sensitive fluorescence detection of fluorophore markers with monitoring of their dark triplet state transitions, allowing imaging of such protein-lipid interactions. We first determined the dark state kinetics of the biomembrane fluorophore 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD) in lipid vesicles, and how its triplet state is quenched by spin-labels in the same membranes. We then monitored collisional quenching of NBD-lipid derivatives by spin-labelled stearic acids in live cell plasma membranes, and of NBD-lipid derivatives by spin-labelled G-Protein Coupled Receptors (GPCRs). We could then resolve transient interactions between the GPCRs and different lipids, how these interactions changed upon GPCR activation, thereby demonstrating a widely applicable means to image and characterize transient molecular interactions in live cell membranes in general, not within reach via traditional fluorescence readouts.

Rhabdoid tumors (RT) are among the most aggressive tumors in early childhood. Overall survival remains poor, and treatment only effectively occurs at the cost of high toxicity and late adverse effects. It has been reported that the neurokinin-1 receptor/ substance P complex plays an important role in cancer and proved to be a promising target. However, its role in RT has not yet been described. This study aims to determine whether the neurokinin-1 receptor is expressed in RT and whether neurokinin-1 receptor (NK1R) antagonists can serve as a novel therapeutic approach in treating RTs. By in silico analysis using the cBio Cancer Genomics Portal we found that RTs highly express neurokinin-1 receptor. We confirmed these results by RT-PCR in both tumor cell lines and in human tissue samples of various affected organs. We demonstrated a growth inhibitory and apoptotic effect of aprepitant in viability assays and flow cytometry. Furthermore, this effect proved to remain when used in combination with the cytostatic cisplatin. Western blot analysis showed an upregulation of apoptotic signaling pathways in rhabdoid tumors when treated with aprepitant. Overall, our findings suggest that NK1R may be a promising target for the treatment of RT in combination with other anti-cancer therapies and can be targeted with the NK1R antagonist aprepitant.

TRAAK (KCNK4, K2P4.1) is a mechanosensitive two-pore domain potassium (K2P) channel. Due to its expression within sensory neurons and genetic link to neuropathic pain it represents a promising potential target for novel analgesics. In common with many other channels in the wider K2P sub-family, there remains a paucity of small molecule pharmacological tools. Specifically, there is a lack of molecules selective for TRAAK over the other members of the TREK subfamily of K2P channels. We developed a thallium flux assay to allow high throughput screening of compounds and facilitate the identification of novel TRAAK activators. Using a library of ∼1200 drug like molecules we identified Aprepitant as a small molecule activator of TRAAK. Aprepitant is an NK-1 antagonist used to treat nausea and vomiting. Close structural analogues of Aprepitant and a range of NK-1 antagonists were also selected or designed for purchase or brief chemical synthesis and screened for their ability to activate TRAAK. Electrophysiology experiments confirmed that Aprepitant activates both the 'long' and 'short' transcript variants of TRAAK. We also demonstrated that Aprepitant is selective and does not activate other members of the K2P superfamily. This work describes the development of a high throughput assay to identify potential TRAAK activators and subsequent identification and confirmation of the novel TRAAK activator Aprepitant. This discovery identifies a useful tool compound which can be used to further probe the function of TRAAK K2P channels.

Locoregionally administered, NK1 receptor (NK1R) targeted radionuclide therapy is a promising strategy for the treatment of glioblastoma multiforme. So far, the radiopharmaceuticals used in this approach have been based on the endogenous agonist of NK1R, Substance P or on its close analogues. Herein, we used a well-known, small molecular NK1R antagonist, L732,138, as the basis for the radiopharmaceutical vector. First, 14 analogues of this compound were evaluated to check whether extending the parent structure with linkers of different lengths would not deteriorate the NK1R binding. The tested analogues had affinity similar to or better than the parent compound, and none of the linkers had a negative impact on the binding. Next, five DOTA conjugates were synthesized and used for labelling with

Substance P binds to the Neurokinin-1 (NK-1) receptors found in the emetic center of the central nervous system (CNS) to induce emesis. Maropitant is a selective NK-1 receptor antagonist that inhibits the binding of substance P to NK-1 receptors and is commonly used to prevent and treat vomiting in dogs. This review study aimed to discuss and analyze the therapeutic potential of substance P (Neurokinin-1 receptor) antagonist with a particular focus on the drug maropitant in canine medicine. A systematic literature review was performed to identify the existing literature on the subject during the past 20 years (2001-2021) using such databases as ScienceDirect, PubMed, Scopus, and Google Scholar. The initial search identified 173 articles; however, 41 articles were selected for further analysis, based on the specific inclusion and exclusion criteria. Studies have already confirmed the role of substance P and NK-1 receptors in central pain processing, intestinal smooth muscle contraction, vasodilation, and neurogenic inflammation. Maropitant is one of the most effective veterinary antiemetic drugs that work well against peripheral and central stimuli that trigger the vomiting center. It has been already demonstrated that the therapeutic efficacy of maropitant for managing acute vomiting in dogs is associated with pancreatitis, gastritis, and parvoviral enteritis. It can also prevent and treat chemotherapy-induced emesis and delay the signs of nausea and adverse gastrointestinal effects. Regarding the broad-spectrum antiemetic activity of maropitant, it can be recommended for managing uremic vomiting in dogs. In addition, it has also exhibited an anesthetic sparing effect since the dogs treated with maropitant require a slightly lower percentage of isoflurane as an inhalational anesthetic. The NK-1 receptors are also identified in different areas of the pain pathways. Therefore, NK-1 receptor antagonists might be effective for managing visceral pain. However, further studies are required to establish the broad therapeutic potential of NK-1 receptor antagonist drugs, such as maropitant in canine medicine. It has been shown that the pain associated with the subcutaneous administration of maropitant is due to metacresol, a preservative used in some formulations. Therefore, the side effects can be eliminated by developing novel maropitant formulations specifically for dogs.

Head and neck cancer is a growing worldwide public health problem, accounting for approximately 1,500,000 new cases and 500,000 deaths annually. Substance P (SP) is a peptide of the tachykinin family, which has roles related to a large number of physiological mechanisms in humans. The implications of SP in carcinogenesis have recently been reported through the stimulation of the neurokinin 1 receptor (NK1R), or directly, through the effects derived from the constitutive activation of NK1R. Consequently, SP/NK1R seems to play relevant roles in cancer, upregulating cell proliferation, cell migration and chronic inflammation, among other oncogenic actions. Furthermore, there is growing evidence pointing to a central role for SP in tumour progression, singularly so in laryngeal and oral squamous cell carcinomas. The current narrative review of the literature focuses on the relationship between the SP/NK1R system and chronic inflammation and cancer in the headand- neck region. We described a role for SP/NK1R in the transition from chronic inflammation of the head and neck mucosa, to preneoplastic and neoplastic transformation and progression.

Oxidative stress which refers to redox imbalance with increased generation of reactive oxygen species (ROS) has been associated with the pathophysiology of diverse disease conditions. Recently, a close, yet not fully understood, relation between oxidative stress and neuropeptides, in particular, substance P (SP), has been reported in certain conditions. SP has been shown to affect the cellular redox environment through activation of neurokinin-1receptor (NK1R). It seems that SP/NK1R system and oxidative stress can act either synergistically or antagonistically in a context-dependent manner, thereby, influencing the pathology of various clinical disorders either destructively or protectively. Importantly, the interactions between oxidative stress and SP/NK1R system can be pharmacologically targeted. Therefore, a better understanding of the redox modulatory properties of SP/NK1R signaling will pave the way for identifying new therapeutic possibilities for attenuating oxidative stress-mediated damage. Towards this end, we performed a comprehensive search through PubMed/Medline and Scopus databases and discussed all related existing literature regarding the interplay between oxidative stress and SP/NK1R system as well as their implication in various clinical disorders, to provide a clear view and hence better management of oxidative damage.

Expression and immunolocalization of Substance P (SP)/Neurokinin-1 Receptor (NK-1R) in breast carcinoma (BC) patients and its association with routine proliferative markers (ER, PR, HER2/neu, and Ki-67) were evaluated. A cross-sectional study was performed on 34 cases of BC. There were 23 cases of group A (grade III), 8 of group B (grade II), and only 3 cases of group C (grade I). All samples were then processed for SP and NK-1R immunohistochemistry for few cases. 14/23 cases (61%) of group A, 7/8 cases (88%) of group B, and 2/3 (67%) cases of group C were SP positive. Overall, strong staining (≥10% tumor cells), labeled as “+3,” was observed in 9/14 (64.2%) cases of group A and 1/8 (12.5%) cases of group B. Moderate staining labelled as “+2” (in ≥10% tumor cells) was observed in 3/14 (21.4%) cases of group A and 4/8 (50%) cases of group B. Weak positive staining “+1” was observed in only 2/14 (14.28%) cases of group A, 2/8 (25%) cases of group B, and all 2/2 (100%) cases of group C. SP and NK-1R are overexpressed in breast carcinomas, and there is significant association between the grade of tumor and their overexpression.

Substance P (SP), a product of the tachykinin 1 (Tac1) gene, is expressed in many hypothalamic neurons. Its wake-promoting potential could be mediated through histaminergic (HA) neurons of the tuberomamillary nucleus (TMN), where functional expression of neurokinin receptors (NKRs) waits to be characterized. As in the process of nociception in the peripheral nervous system (PNS) capsaicin-receptor (transient potential vanilloid 1: TRPV1) signalling is amplified by local release of histamine and SP, we tested the involvement of tachykinins in the capsaicin-induced long-lasting enhancement (LLEcaps) of HA neurons firing by investigating selective neurokinin receptor ligands in the hypothalamic mouse brain slice preparation using patch-clamp recordings in cell-attached mode combined with single-cell RT-PCR. We report that the majority of HA neurons respond to SP (EC50 3 nM), express the SP precursor tachykinin 1 (Tac1) gene and at least one of the neurokinin receptors. Responses to selective agonists of three known neurokinin receptors were sensitive to corresponding antagonists. LLEcaps was significantly impaired by the neurokinin receptor antagonists, indicating that in hypothalamus, as in the PNS, release of tachykinins downstream to TRPV1 activation is able to boost the release of histamine. The excitatory action of SP on histaminergic neurons adds another pathway to the noradrenergic and orexinergic ones to synergistically enhance cortical arousal. We show NK1R to play a prominent role on HA neurons and thus the control of wakefulness.

The basal ganglia are a group of sub-cortical structures believed to play a critical role in action selection and sequencing. The striatum is the largest input structure of the basal ganglia and contains the neuropeptide substance P in abundance. Recent computational work has suggested that substance P could play a critical role in action sequence performance and acquisition, but this has not been tested experimentally before. The aim of the present study was to test how blocking substance P's main NK1-type receptors affected the sequential and temporal organization of spontaneous behavioral patterns. We did this in rats by focusing on the grooming chain, an innate and highly stereotyped ordered sequence. We performed an open field experiment in which the NK1 receptor antagonist L-733,060 was injected intraperitoneally in rats at two doses (2 and 4 mg/kg/ml), in a within-subject counterbalanced design. We used first order transition probabilities, Variable Length Markov Models, entropy metrics and T-pattern analysis to evaluate the effects of L-733,060 on sequential and temporal aspects of spontaneously ordered behavioral sequences. Our results suggest that blocking NK1 receptors made the transitions between the grooming chain elements significantly more variable, the transition structure of the grooming bouts simpler, and it increased the probability of transitioning from active to inactive states. Overall, this suggest that blocking substance P receptors led to a general break down in the fluency of spontaneous behavioral sequences, suggesting that substance P could be playing a key role in the implementation of sequential patterns.

Opioid agonists are well-established analgesics, widely prescribed for acute but also chronic pain. However, their efficiency comes with the price of drastically impacting side effects that are inherently linked to their prolonged use. To answer these liabilities, designed multiple ligands (DMLs) offer a promising strategy by co-targeting opioid and non-opioid signaling pathways involved in nociception. Despite being intimately linked to the Substance P (SP)/neurokinin 1 (NK1) system, which is broadly examined for pain treatment, the neurokinin receptors NK2 and NK3 have so far been neglected in such DMLs. Herein, a series of newly designed opioid agonist-NK2 or -NK3 antagonists is reported. A selection of reported peptidic, pseudo-peptidic, and non-peptide neurokinin NK2 and NK3 ligands were covalently linked to the peptidic μ-opioid selective pharmacophore Dmt-DALDA (H-Dmt-d-Arg-Phe-Lys-NH2) and the dual μ/δ opioid agonist H-Dmt-d-Arg-Aba-βAla-NH2 (KGOP01). Opioid binding assays unequivocally demonstrated that only hybrids SBL-OPNK-5, SBL-OPNK-7 and SBL-OPNK-9, bearing the KGOP01 scaffold, conserved nanomolar range μ-opioid receptor (MOR) affinity, and slightly reduced affinity for the δ-opioid receptor (DOR). Moreover, NK binding experiments proved that compounds SBL-OPNK-5, SBL-OPNK-7, and SBL-OPNK-9 exhibited (sub)nanomolar binding affinity for NK2 and NK3, opening promising opportunities for the design of next-generation opioid hybrids.

Prostate cancer continues to be one of the main global health issues in men. Neuropeptide substance P (SP) acting via neurokinin-1receptor (NK1R) promotes tumorigenicity in many human malignant tumors. However, its pro-tumorigenic functions and the therapeutic effects of its inhibition in prostate cancer remain unclear.MTT assay was employed for measuring cellular proliferation and cytotoxicity. mRNAs and proteins expression levels were evaluated by qRT-PCR and western blot assay, respectively. Gelatinase activity was assessed by zymography. The migration ability was defined using wound-healing assay. Flow cytometry was employed to evaluate the cell cycle distribution. We also performed an in vivo experiment in a mouse model of prostate cancer to confirm the in vitro therapeutic effect of targeting the SP/NK1R system.We found a noticeable increase in the expression of the truncated isoform of NK1R as an oncogenic NK1R splice variant in tumor cells. We also demonstrated that SP promotes both proliferative and migrative phenotypes of prostate cancer through modifying cell cycle-related proteins (c-Myc, cyclin D1, cyclin B1, p21), and apoptosis-related genes (Bcl-2 and Bax), promoting cell migration and increasing MMP-2 and MMP-9 expression and activity, while aprepitant administration could remarkably reverse these effects. SP also stimulated tumor growth in vivo, which was correlated with shorter survival times, while aprepitant reversed this effect and led to significantly longer survival time.Our findings suggest that SP/NK1R system may serve as a novel therapeutic target in prostate cancer and support the possible candidacy of aprepitant in future prostate cancer therapy.

Until recently, itch pathophysiology was poorly understood and treatments were poorly effective in relieving itch. Current progress in our knowledge of the itch processing, the numerous mediators and receptors involved has led to a large variety of possible therapeutic pathways. Currently, inhibitors of IL-31, IL-4/13, NK1 receptors, opioids and cannabinoids, JAK, PDE4 or TRP are the main compounds involved in clinical trials. However, many new targets, such as Mas-related GPCRs and unexpected new pathways need to be also explored.

Background. Glioblastoma or glioma is the most common malignant brain tumor. Patients have a prognosis of approximately 15 months, despite the current aggressive treatment. Neurokinin-1 receptor (NK-1R) occurs naturally in human glioma, and it is necessary for the tumor development. Objective. The purpose of the study was to increase the knowledge about the involvement of the substance P (SP)/NK-1R system in human glioma. Methods. Cellular localization of NK-1R and SP was studied in GAMG and U-87 MG glioma cell lines by immunofluorescence. The contribution of both SP and NK-1R to the viability of these cells was also assessed after applying the tachykinin 1 receptor (TAC1R) or the tachykinin 1 (TAC1) small interfering RNA gene silencing method, respectively. Results. Both SP and the NK-1R (full-length and truncated isoforms) were localized in the nucleus and cytoplasm of GAMG and U-87 MG glioma cells. The presence of full-length NK-1R isoform was mainly observed in the nucleus, while the level of truncated isoform was higher in the cytoplasm. Cell proliferation was decreased when glioma cells were transfected with TAC1R siRNA, but not with TAC1. U-87 MG cells were more sensitive to the effect of the TAC1R inhibition than GAMG cells. The decrease in the number of glioma cells after silencing of the TAC1R siRNA gene was due to apoptotic and necrotic mechanisms. In human primary fibroblast cultured cells, TAC1R silencing by siRNA did not produce any change in cell viability. Conclusions. Our results show for the first time that the expression of the TAC1R gene (NK-1R) is essential for the viability of GAMG and U-87 MG glioma cells. On the contrary, the TAC1R gene is not essential for the viability of normal cells, confirming that NK-1R could be a promising and specific therapeutic target for the treatment of glioma. Junta de Andalucía BIO-158

Although there is no doubt regarding the involvement of oxidative stress in the development of glioblastoma, many questions remained unanswered about signaling cascades that regulate the redox status. Given the importance of the substance P (SP)/neurokinin 1 receptor (NK1R) system in different cancers, it was of particular interest to evaluate whether the stimulation of this cascade in glioblastoma-derived U87 cells is associated with the induction of oxidative stress. Our results showed that SP-mediated activation of NK1R not only increased the intracellular levels of malondialdehyde (MDA) and reactive oxygen species (ROS) but also reduced the concentration of thiol in U87 cells. We also found that upon SP addition, there was a significant reduction in the cells’ total antioxidant capacity (TAC), revealing that the SP/NK1R axis may be involved in the regulation of oxidative stress in glioblastoma cells. The significant role of SP/NK1R in triggering oxidative stress in glioblastoma has become more evident when we found that the abrogation of the axis using aprepitant reduced cell survival, probably through exerting antioxidant effects. The results showed that both MDA and ROS concentrations were significantly reduced in the presence of aprepitant, and the number of antioxidant components of the redox system increased. Overall, these findings suggest that aprepitant might exert its anticancer effect on U87 cells through shifting the balance of oxidant and antioxidant components of the redox system.

Gene therapy provides a promising treatment for glioblastoma multiforme, which mainly depends on two key aspects, crossing the blood brain barrier (BBB) effectively and transfecting target cells selectively. In this work, we reported a series of peptide-based vectors for transfecting glioma cells specifically consisting of several functional segments including a cell-penetrating peptide, targeting segment substance P (SP), an endosomal escape segment, a PEG linker and a stearyl moiety. The conformations and DNA-loading capacities of peptide vectors and the self-assembly behaviors of peptide/pGL3 complexes were characterized. The in vitro gene transfection was evaluated in U87, 293T-NK1R, and normal 293T cell lines. The transfection efficiency ratio of P-02 (SP-PEG4-K(C18)-(LLHH)3-R9) to Lipo2000 in the U87 cell line was about 36% higher than that in the 293T cell line. The neurokinin-1 receptor (NK1R) in U87 cells mediated the transfection process via interactions with the ligand SP in peptide vectors. The mechanism of NK1R mediated transfection was demonstrated by the use of gene-modified 293T cells expressing NK1R, as well as the gene transfection in the presence of free SP. Besides, P-02 could promote the pGL3 plasmids to cross the BBB model in vitro and achieved the EGFP gene transfection in the brain of zebrafish successfully. The designed peptide vectors, owing to their specific transfection capacity in glioma cells, provide a potential approach for glioblastoma multiforme gene therapy.

Hematoma clearance is an important therapeutic target to improve outcome following intracerebral hemorrhage (ICH). Recent studies showed that Neurokinin receptor-1 (NK1R) inhibition exerts protective effects in various neurological disease models, but its role in ICH has not been explored. The objective of this study was to investigate the role of NK1R and its relation to hematoma clearance after ICH using an autologous blood injection mouse model. A total of 332 adult male CD1 mice were used. We found that the expression levels of NK1R and its endogenous ligand, substance P (SP), were significantly upregulated after ICH. Intraperitoneal administration of the NK1R selective antagonist, Aprepitant, significantly improved neurobehavior, reduced hematoma volume and hemoglobin levels after ICH, and promoted microglia polarization towards M2 phenotype. Aprepitant decreased phosphorylated PKC, p38MAPK, and NFκB p65, and downregulated M1 markers while upregulating M2 markers after ICH. Intracerebroventricular administration of the NK1R agonist, GR73632 or PKC agonist, phorbol 12-myristate 13-acetate (PMA) reversed the effects of Aprepitant. To demonstrate the upstream mediator of NK1R activation, we performed thrombin injection and found that it increased SP. Inhibiting thrombin suppressed SP and decreased M1 markers while increasing M2 microglia polarization. Thus, NK1R inhibition promoted hematoma clearance after ICH by increasing M2 microglial polarization via downregulating PKC/p38MAPK/NFκB signaling pathway, and thrombin may be a key upstream mediator of NK1R activation. Therapeutic interventions inhibiting NK1R signaling may be a new target for the treatment of ICH. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13311-021-01077-8.

A remarkable molecular and functional heterogeneity of the primary sensory neurons and dorsal horn interneurons transmits pain- and or itch-relevant information, but the molecular signature of the projection neurons that convey the messages to the brain is unclear. Here, using retro-TRAP (translating ribosome affinity purification) and RNA-seq we reveal extensive molecular diversity of spino- and trigeminoparabrachial projection neurons, which to date are almost exclusively defined by their expression of the neurokinin 1 receptor (NK1R). Among the many genes identified, we highlight distinct subsets of Cck+, Nptx2+, Nmb+, and Crh+ expressing projection neurons. By combining in situ hybridization of retrogradely labeled neurons with Fos-based assays we also demonstrate significant functional heterogeneity, including both convergence and segregation of pain- and itch-provoking inputs onto molecularly diverse subsets of NK1R- and non-NK1R-expressing projection neurons. The current study provides the first comprehensive investigation into the molecular profiles and functional properties of projection neuron subtypes.

BACKGROUND: Allergic contact dermatitis (CD) is a chronic inflammatory skin disease caused by type-1 biased adaptive immunity for which there is an unmet need for antigen (Ag)-specific immunotherapies. Exposure to skin sensitizers stimulates secretion of the proinflammatory neuropeptides substance P (SP) and hemokinin 1 (HK1), which signal via the neurokinin-1 receptor (NK1R) to promote the innate and adaptive immune responses of CD. Accordingly, mice lacking the NK1R develop impaired CD. Nonetheless, the role and therapeutic opportunities of targeting the NK1R in CD remain to be elucidated. OBJECTIVE: To develop an Ag-specific immunosuppressive approach to treat CD by skin co-delivery of hapten and NK1R antagonists integrated in dissolvable microneedle arrays (MNA). METHODS: In vivo mouse models of contact hypersensitivity and ex-vivo models of human skin were used to delineate the effects and mechanisms of NK1R-signaling and the immunosuppressive effects of the contact sensitizer-NK1R-antagonists-MNA in CD. RESULTS: We demonstrate in mice that CD requires NK1R signaling by SP and HK1. Specific deletion of the NK1R in keratinocytes and dendritic cells, but not in mast cells prevented CD. Skin co-delivery of hapten- or Ag-NK1R-antagonist-MNA inhibited neuropeptide-mediated skin inflammation in mouse and human skin, promoted deletion of Ag-specific effector T cells and increased regulatory T cells, which prevented CD onset and relapses locally and systemically in an Ag-specific manner. CONCLUSIONS: Our findings demonstrate that immune-regulation by engineering localized skin neuroimmune networks can be used to treat cutaneous diseases that like CD, are caused by type-1 immunity.

The antagonists of the neurokinin-1 receptor (NK1R) are known for their anti-inflammatory, anxiolytic, antiemetic, and anticancer activities. Aprepitant, a nonpeptide NK1R antagonist, is used in nausea and vomiting, the most common side effects of cancer chemotherapy in patients. It has been established that NK1R activation by substance P (SP), which links cancer promotion and progression to a neurokinin-mediated environment, became one mechanism that corresponds to the mitogenesis of tumor cells. Therefore, this study is aimed at explaining and evaluating the anticancer impacts of aprepitant on esophageal squamous cancer cell (ESCC) spheres by using in vitro experiments, such as resazurin, ROS, annexin-V binding, RT-PCR, and Western blot analysis. As a result, we showed that aprepitant had strong antiproliferative and cytotoxic effects on ESCC cell spheres. Also, aprepitant caused significant G2-M cell cycle arrest depending on concentration increase. Further, exposure of cells to this agent resulted in caspase -8/-9-dependent apoptotic pathway activation by modifying the expression of genes involved in apoptosis. Besides, treatment of the cells by aprepitant abrogates of the PI3K/Akt pathway, as shown by reducing the level of Akt, induces apoptotic cell death. In summary, pharmacological inhibition of NK1R with aprepitant seems to have a significant chance of treating ESCC as a single agent or in conjunction with other chemotherapeutic drugs.

Background Colorectal cancer (CRC) is recognized as one of the most common malignancies with a high mortality rate worldwide, supporting the necessity for an effective novel antitumor drug to improve current therapy's effectiveness. Substance P (SP) is the essential member of the tachykinins (TKs) family, which binds to the specific receptors, known as neurokinin-1 receptor (NK1R), exerting its multiple influences such as tumor cell proliferation, angiogenesis, and metastasis. Aprepitant, as a specific NK1R antagonist, is suggested as a novel antitumor agent, promoting apoptotic processes in tumor cells; however, the exact antitumor mechanism of aprepitant on molecular signaling in CRC is not entirely known. Method The resazurin assay was conducted to assess the cytotoxic effects of aprepitant on the viability of the CRC cell line (SW480). The level of reactive oxygen species (ROS) was measured after 24-hour treatment with SP and aprepitant. PI/annexin V-FITC staining was conducted to assess apoptosis. Also, the expression of NF-κB antiapoptotic target genes and proapoptotic p53 target genes was measured by real-time- (RT-) PCR assay. Western blotting assay was performed to determine the expression of PI3k/AKT/NF-κB proteins. Results We found that aprepitant stimulates apoptotic cell death and attenuates the PI3K/Akt pathway and its downstream proapoptotic target gene, including NF-κB in SW480 cells. Also, the obtained results from the quantitative RT-PCR assay showed that aprepitant could decrease the level of mRNA of NF-κB antiapoptotic target genes. Conclusion Towards this end, this study suggests that SP/NK1R system plays a vital role in the development of CRC, and pharmaceutical targeting of NK1R using aprepitant might be a promising treatment against CRC.

Pathological impulsivity is a debilitating symptom of multiple psychiatric diseases with few effective treatment options. To identify druggable receptors with anti-impulsive action we developed a systematic target discovery approach combining behavioural chemogenetics and gene expression analysis. Spatially restricted inhibition of three subdivisions of the prefrontal cortex of mice revealed that the anterior cingulate cortex (ACC) regulates premature responding, a form of motor impulsivity. Probing three G-protein cascades with designer receptors, we found that the activation of Gi-signalling in layer-5 pyramidal cells (L5-PCs) of the ACC strongly, reproducibly, and selectively decreased challenge-induced impulsivity. Differential gene expression analysis across murine ACC cell-types and 402 GPCRs revealed that - among Gi-coupled receptor-encoding genes - Grm2 is the most selectively expressed in L5-PCs while alternative targets were scarce. Validating our approach, we confirmed that mGluR2 activation reduced premature responding. These results suggest Gi-coupled receptors in ACC L5-PCs as therapeutic targets for impulse control disorders., publishedVersion

Pax6 is an important transcription factor expressed in several discrete domains of the developing central nervous system. It has been reported that Pax6 is involved in the specification of subtypes of hindbrain motor neurons. Pax6 homozygous mutant (rSey2/rSey2) rats die soon after birth, probably due to impaired respiratory movement. To determine whether the respiratory center in the medulla functions normally, we analyzed the histological and neurophysiological properties of the medulla and spinal cord in fetal rats with this mutation. First, the medulla of rSey2/rSey2 at embryonic (E) 21.5-E22.5 tended to be smaller than those from heterozygous mutant (rSey2/+) and wild-type (+/+) littermates. Through immunohistochemical analysis, we confirmed normal distribution of Phox2b-expressing cells in the parafacial respiratory group (pFRG) of rSey2/rSey2 rats. Expression of neurokinin-1 receptor (NK-1R) was weak and dispersed in rSey2/rSey2 rats. In addition, rSey2/rSey2 rats have a defect of the hypoglossal nerve root. Electrophysiological analysis using brainstem-spinal cord preparations (E21.5-E22.5) revealed that rSey2/rSey2 rats showed larger fluctuation of the amplitude of inspiratory activity monitored from the fourth cervical root although there was no significant difference in the respiratory rate among rSey2/rSey2, rSey2/+, and +/+ littermates. The response of respiratory rhythm to high CO2 was similar among all genotypes. Optical recordings of neuronal activity revealed that the activity of the pFRG tended to be weaker and inspiratory activity appeared in more scattered areas in the caudal ventral medulla in the rSey2/rSey2 rats. These results suggest that the basal activity of the respiratory system was preserved with mild impairment of the inspiratory activity in the rSey2/rSey2 rats and that the Pax6 gene is involved in the functional development of the neuronal system producing effective inspiratory motor outputs for survival.

Purpose: To investigate the influence of unilateral ultraviolet radiation (UVR) exposure on the unexposed, partner eye in vivo. To characterize the immunological cross-talk between the eyes and verify a sympathizing reaction of the partner eye via a neurokinin-dependent signaling pathway of substance P and its neurokinin-1 receptor (NKR-1) and/or monocyte chemoattractant protein-1 (MCP-1). Methods: C57BL/6 mice were unilaterally exposed in vivo to UVR-B to a 5-fold cataract threshold equivalent dose of 14.5 kJ/m2 with a UV irradiation Bio-Spectra system. The unexposed contralateral eye was completely shielded during irradiation. After 3 and 7 days post exposure, eyes were stained with fluorescence-coupled antibody for substance P NKR-1. The same was performed in control animals receiving only anesthesia but no UVR-B exposure. NKR-1 and MCP-1 levels in ocular tissue lysates were quantified by enzyme-linked immunosorbent assay. Results: UVR-B induces NKR-1 upregulation after 3 and 7 days in the exposed and in the unexposed, contralateral mouse eye. NKR-1 protein level was upregulated in the exposed and contralateral iris/ciliary body complex, choroidea and in the contralateral retina as well as in the exposed cornea. MCP-1 levels were elevated in the exposed cornea, iris/ciliary body complex, and aqueous humor but not in contralateral ocular tissues. Conclusions: UVR-B exposure triggers NKR-1 upregulation not only in the exposed but also in the unexposed, partner eye in various ocular tissues. Following UVR-B exposure, MCP-1 protein levels are upregulated in the exposed eye, but the contralateral side remains unaffected.

Neuroendocrine tumors (NETs) have reasonably high 5-year survival rates when diagnosed at an early stage but are significantly more lethal when discovered only after metastasis. Although several imaging modalities such as computed tomography (CT), positron emission tomography, and magnetic resonance imaging can detect neuroendocrine tumors, their high false positive rates suggest that more specific diagnostic tests are required. Targeted imaging agents such as Octreoscan® have met some of this need for improved specificity, but their inability to image poorly differentiated NETs suggests that improved NET imaging agents are still needed. Because neurokinin 1 receptors (NK1Rs) are widely over-expressed in neuroendocrine tumors, but show limited expression in healthy tissues, we have undertaken to develop an NK1R-targeted imaging agent for improved diagnosis and staging of neuroendocrine tumors. A small molecule NK1R antagonist was conjugated via a flexible spacer to a Tc-99m chelating peptide. After complexation with Tc-99m, binding of the conjugate to human embryonic kidney (HEK293) cells transfected with the human NK1R was evaluated as a function of radioimaging agent concentration. In vivo imaging of HEK293-NK1R tumor xenografts in mice was also performed by single-photon emission computed tomography/computed tomography (γ-SPECT/CT), and the distribution of the conjugate in various tissues was quantified by tissue resection and γ-counting. NK1R-targeted Tc-99m-based radioimaging agent displayed excellent affinity (Kd = 16.8 nM) and specificity for HEK293-NK1R tumor xenograft. SPECT/CT analysis of tumor-bearing mice demonstrated significant tumor uptake and high tumor to background ratio as early as 2 h post injection. The excellent tumor contrast afforded by our NK1R-targeted radioimaging agent exhibits properties that could improve early diagnosis and staging of many neuroendocrine tumors.

Cardiac fibrosis is an underlying cause of diastolic dysfunction, contributing to heart failure. Substance P (SP) activation of the neurokinin-1 receptor (NK-1R) contributes to cardiac fibrosis in hypertension. However, based on in vitro experiments, this does not appear to be via direct activation of cardiac fibroblasts. While numerous cells could mediate the fibrotic effects of SP, herein, we investigate mast cells (MC) as a mechanism mediating the fibrotic actions of SP, since MCs are known to play a role in cardiac fibrosis and respond to SP. Spontaneously hypertensive rats (SHR) were treated with the NK-1R antagonist L732138 (5 mg/kg/d) from 8 to 12 weeks of age. L732138 prevented increased MC maturation of resident immature MCs. NK-1R blockade also prevented increased cardiac MC maturation in angiotensin II-infused mice. MC-deficient mice were used to test the importance of MC NK-1Rs to MC activation. MC-deficient mice administered angiotensin II did not develop fibrosis; MC-deficient mice reconstituted with MCs did develop fibrosis. MC-deficient mice reconstituted with MCs lacking the NK-1R also developed fibrosis, indicating that NK-1Rs are not required for MC activation in this setting. In conclusion, the NK-1R causes MC maturation, however, other stimuli are required to activate MCs to cause fibrosis.

Background Chronic pruritus is a distressing symptom associated with various dermatological conditions and systemic diseases. Current treatment options are often inadequate, resulting in impaired quality of life for many patients. An understanding of the underlying mechanisms of itch across pruritic conditions is important for development of effective, targeted treatments for chronic pruritus. Objectives To provide an overview of the pathogenesis of chronic pruritus, focusing on the role of substance P (SP) and neurokinin 1 receptor (NK1 R) in itch signalling, and to describe data supporting NK1 R antagonism as a potential strategy for the treatment of chronic pruritus. Methods A PubMed search was conducted to determine what data were available that investigated the role of SP and NK1 R in itch signalling. Results SP is a neuropeptide that is a mediator of itch signalling. One of the target receptors for SP is NK1 R, which is expressed in the central nervous system and on multiple cell types involved in the initiation and transmission of itch. Studies demonstrating that SP and NK1 R are overexpressed across multiple chronic itch-inducing conditions and that NK1 R antagonism disrupts itch signalling and reduces itch provide a rationale for targeting this pathway as a potential treatment of chronic pruritus across multiple diseases. Conclusions A large and growing body of evidence, including recent phase II clinical studies of NK1 R antagonists, demonstrate that SP and NK1 R play an important role in itch signalling. Additional studies are ongoing to further evaluate the use of NK1 R antagonists for the treatment of chronic pruritus. What's already known about this topic? Chronic pruritus has a significant impact on quality of life. Current treatment options for chronic pruritus are inadequate. Substance P (SP) and neurokinin 1 receptor (NK1 R) have been shown to play a role in itch signalling, and may be a rational target for addressing chronic pruritus. NK1 R antagonists are being evaluated as potential treatment for chronic pruritus. What does this study add? This review provides a compilation of the most up-to-date data elucidating the role of SP and NK1 R in itch signalling, which supports targeting this pathway as a potential treatment of chronic pruritus. NK1 R antagonism disrupts itch signalling and reduces itch. A summary of the latest data on NK1 R antagonists in the treatment of pruritus is provided.

Background:In adults, the most lethal and frequent primary brain tumor is glioblastoma. Despite multimodal aggressive therapies, the median survival time after diagnosis is around 15 months. In part, this is due to the blood-brain barrier that restricts common treatments (e.g., chemotherapy). Unfortunately, glioma recurs in 90% of patients. New therapeutic strategies against glioma are urgently required. Substance P (SP), through the neurokinin (NK)-1 receptor, controls cancer cell proliferation by activating c-myc, mitogenactivated protein kinases, activator protein 1 and extracellular signal-regulated kinases 1 and 2. Glioma cells overexpress NK-1 receptors when compared with normal cells. The NK-1 receptor/SP system regulates the proliferation/migration of glioma cells and stimulates angiogenesis, triggering inflammation which contributes to glioma progression. In glioma cells, SP favors glycogen breakdown, essential for glycolysis. By contrast, in glioma, NK-1 receptor antagonists block the proliferation of tumor cells and the breakdown of glycogen and also promote the death (apoptosis) of these cells. These antagonists also inhibit angiogenesis and exert antimetastatic and anti-inflammatory actions.Objective:This review updates the involvement of the NK-1 receptor/SP system in the development of glioma and the potential clinical application of NK-1 receptor antagonists as antiglioma agents.Conclusion:The NK-1 receptor plays a crucial role in glioma and NK-1 receptor antagonists could be used as anti-glioma drugs.

Chronic inflammation in fibroblast-like synoviocytes (FLSs) induced by pro-inflammatory cytokines such as TNF-α plays a key role in the pathogenesis of rheumatoid arthritis (RA). The neurokinin-1 receptor (NK-1R) is one of the G protein-coupled receptors (GPCRs) mediating the intracellular signalling of substance P (SP). However, the possible implications of NK-1R in rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs) and the pathogenesis of RA have not yet been reported. In the current study, we report that NK-1R is expressed in FLSs. Importantly, NK-1R expression was found to be significantly increased in RA-FLSs compared to normal FLSs. Interestingly, we found that treatment with tumour necrosis factor (TNF)-α increased the expression of NK-1R at both the gene and protein levels. Treatment with the NK-1R antagonist aprepitant reduced TNF-α-induced expression of NADPH oxidase 4 (NOX-4) and generation of reactive oxygen species (ROS) in FLSs. Our results also display that blockage of NF-1R using aprepitant inhibited TNF-α-induced expression and secretion of proinflammatory cytokines, including interleukin-1β (IL-1β), IL-6, and IL-8. Consistently, aprepitant prevented TNF-α-induced expression of matrix metalloproteinases (MMPs), including matrix metalloproteinase-3 (MMP-3) and matrix metalloproteinase-13 (MMP-13). Mechanistically, our data demonstrate that treatment with aprepitant inhibited TNF-α-induced phosphorylation and degradation of inhibitor of NF-κB (IκBα). Notably, aprepitant attenuated TNF-α-induced nuclear translocation of nuclear factor κB (NF-κB) p65 and reduced luciferase activity of NF-κB in FLSs. The findings implicated a novel function of NK-1R in RA-FLSs. Blockage of NK-1R using its specific antagonist aprepitant might provide a new therapeutic strategy for RA.

The medial habenula-interpeduncular nucleus (MHb-IPN) pathway has recently been implicated in the suppression of fear memory. A notable feature of this pathway is the corelease of neurotransmitters and neuropeptides from MHb neurons. Our studies in mice reveal that an activation of substance P-positive dorsomedial habenula (dMHb) neurons results in simultaneous release of glutamate and glycine in the lateral interpeduncular nucleus (LIPN). This glycine receptor activity inhibits an activity-dependent long-lasting potentiation of glutamatergic synapses in LIPN neurons, while substance P enhances this plasticity. An endocannabinoid CB1 receptor-mediated suppression of GABA(B) receptor activity allows substance P to induce a long-lasting increase in glutamate release in LIPN neurons. Consistent with the substance P-dependent synaptic potentiation in the LIPN, the NK1R in the IPN is involved in fear extinction but not fear conditioning. Thus, our study describes a novel plasticity mechanism in the LIPN and a region-specific role of substance P in fear extinction.

Hybrid compounds (also known as chimeras, designed multiple ligands, bivalent compounds) are chemical units where two active components, usually possessing affinity and selectivity for distinct molecular targets, are combined as a single chemical entity. The rationale for using a chimeric approach is well documented as such novel drugs are characterized by their enhanced enzymatic stability and biological activity. This allows their use at lower concentrations, increasing their safety profile, particularly when considering undesirable side effects. In the group of synthetic bivalent compounds, drugs combining pharmacophores having affinities toward opioid and neurokinin-1 receptors have been extensively studied as potential analgesic drugs. Indeed, substance P is known as a major endogenous modulator of nociception both in the peripheral and central nervous systems. Hence, synthetic peptide fragments showing either agonism or antagonism at neurokinin 1 receptor were both assigned with analgesic properties. However, even though preclinical studies designated neurokinin-1 receptor antagonists as promising analgesics, early clinical studies revealed a lack of efficacy in human. Nevertheless, their molecular combination with enkephalin/endomorphin fragments has been considered as a valuable approach to design putatively promising ligands for the treatment of pain. This paper is aimed at summarizing a 20-year journey to the development of potent analgesic hybrid compounds involving an opioid pharmacophore and devoid of unwanted side effects. Additionally, the legitimacy of considering neurokinin-1 receptor ligands in the design of chimeric drugs is discussed.

Aberrant substance P/neurokinin‐1 receptor (SP/NK‐1R) system activation plays a critical role in various disorders, however, little is known about the expression and the detailed molecular mechanism of the SP and NK‐1R in gallbladder cancer (GBC). In this study, we firstly analyzed the expression and clinical significance of them in patients with GBC. Then, cellular assays were performed to clarify their biological role in GBC cells. Moreover, we investigated the molecular mechanisms regulated by SP/NK‐1R. Meanwhile, mice xenografted with human GBC cells were analyzed regarding the effects of SP/NK1R complex in vivo. Finally, patient samples were utilized to investigate the effect of SP/NK‐1R. The results showed that SP and NK‐1R were highly expressed in GBC. We found that SP strongly induced GBC cell proliferation, clone formation, migration and invasion, whereas antagonizing NK‐1R resulted in the opposite effects. Moreover, SP significantly enhanced the expression of NF‐κB p65 and the tumor‐associated cytokines, while, Akt inhibitor could reverse these effects. Further studies indicated that decreasing activation of NF‐κB or Akt diminished GBC cell proliferation and migration. In consistent with results, immunohistochemical staining showed high levels of Akt, NF‐κB and cytokines in tumor tissues. Most importantly, the similar conclusion was obtained in xenograft mouse model. Our findings demonstrate that NK‐1R, after binding with the endogenous agonist SP, could induce GBC cell migration and spreading via modulation of Akt/NF‐κB pathway.

Mitochondria, as primary energy generators and Ca2+ biosensor, are dynamically coupled to neuronal activities, and thus play a role in neuroplasticity. Here we report that respiratory neuroplasticity induced by daily acute intermittent hypoxia (dAIH) evoked adaptive changes in the ultrastructure and postsynaptic distribution of mitochondria in the pre-Botzinger complex (pre-BotC). The metabolic marker of neuronal activity, cytochrome c oxidase (CO), and dendritic mitochondria were examined in pre-BotC neurons of adult Sprague-Dawley rats preconditioned with dAIH, which is known to induce long-term facilitation (LTF) in respiratory neural activities. We performed neurokinin 1 receptor (NK1R) pre-embedding immunocytochemistry to define pre-BotC neurons, in combination with CO histochemistry, to depict ultrastructural alterations and CO activity in dendritic mitochondria. We found that the dAIH challenge significantly increased CO activity in pre-BotC neurons. Darkly CO-reactive mitochondria at postsynaptic sites in the dAIH group were much more prevalent than those in the normoxic control. In addition, the length and area of mitochondria were significantly increased in the dAIH group, implying a larger surface area of cristae for ATP generation. There was a fine, structural remodeling, notably enlarged and branching mitochondria or tapered mitochondria extending into dendritic spines. Mitochondrial cristae were mainly in parallel-lamellar arrangement, indicating a high efficiency of energy generation. Moreover, flocculent or filament-like elements were noted between the mitochondria and the postsynaptic membrane. These morphological evidences, together with increased CO activity, demonstrate that dendritic mitochondria in the pre-BotC responded dynamically to respiratory plasticity. Hence, plastic neuronal changes are closely coupled to active mitochondrial bioenergetics, leading to enhanced energy production and Ca2+ buffering that may drive the LTF expression.

Purpose: Chemotherapy-induced nausea and vomiting (CINV) is one of the most feared and disturbing adverse events of cancer treatment associated with decreased adherence to effective chemotherapy regimens. For high-risk soft tissue sarcoma patients, receiving multiple-day chemotherapy (MD-CT), antiemetic guidelines recommend a combination of an NK 1 receptor antagonist (NK 1 -RA), a 5-HT 3 receptor antagonist (5HT 3 -RA), and dexamethasone on each day of the antineoplastic treatment. NEPA is the first oral fixed-dose combination of a highly selective NK 1 -RA, netupitant, and second-generation 5HT 3 -RA, palonosetron. So far, no data has been published in literature about the efficacy of a single dose of NEPA in MD-CT. Methods: We performed a prospective, non-comparative study to assess the efficacy of one shot of NEPA plus dexamethasone in sarcoma patients receiving MD-CT. The primary efficacy endpoint was a complete response (CR: no emesis, no rescue medication) during the overall phase (0–120h) in cycle 1. The main secondary endpoints were CR during the overall phase of cycles 2 and 3. Results: The primary endpoint was reached in 88.9% of patients. Cycles 2 and 3 overall CR rates were 88.9% and 82.4%, respectively. The antiemetic regimen was well tolerated. Conclusions: This pilot study showed the benefit of one shot of NEPA to prevent CINV in sarcoma patients receiving MD-chemotherapy.

Substance P (SP) is believed to play a role in traumatic brain injury (TBI), and the inhibition of binding of SP to the tachykinin neurokinin-1 receptor (NK1R) using NK1R antagonists had made favorable effects on TBI. Our current study addresses the functional roles and underlying mechanisms of SP and NK1R antagonist L-733,060 following TBI. Adult male wild type C57BL/6 J and SP knock out (SP KO) mice received a controlled cortical impact and outcome parameters were assessed. The results showed that TBI-induced motor and spatial memory deficits, lesion volume, brain water content and blood-brain barrier disruption were alleviated both in L-733,060-treated C57BL/6 J mice and vehicle-treated SP KO mice. L-733,060 treatment and SP deletion inhibited TBI-induced the release of cytochrome c from mitochondria to cytoplasm, activation of caspase-3, oxidative stress and neuroinflammation. Higher SP levels in serum and cortex were observed in wild type mice undergoing TBI relative to wild type sham group, but very little expression of cortical SP was detected in the SP-/- mice either TBI or not. Upregulation of NK1R expression after TBI was observed, and there was no significant difference between wild type and SP KO groups. in vitro, L-733,060 and SP deletion inhibited scratch injury-induced cell death, loss of mitochondrial membrane potential and reactive oxygen species (ROS) production following TBI. Together, the results of this study implicate a functional role for NK1-R antagonist L-733,060 and deletion of SP in TBI-induced neurological outcome, oxidative damage, neuroinflammation and cell death. Upregulation of NK1R maybe a consequence of TBI, independent of the levels of substance P. This study raises the possibility that targeting SP through its receptor NK1R or genetic deletion may have therapeutic efficacy in TBI.

Atherosclerosis is the main cause of many cardiovascular diseases. Endothelial dysfunction is recognized as an early event in the development of atherosclerosis. Many drugs have been studied to mitigate hyperlipidemia-induced endothelial injury. Studies have demonstrated that neuropeptide substance P (SP) and its preferred receptor neurokinin receptor 1 (NK-1R) are involved in the pathological progression of cardiovascular disease. In this study, we show that aprepitant, a selective NK-1R antagonist, possesses beneficial effects that protect endothelial cells from oxidized low-density lipoprotein (ox-LDL)-induced inflammatory response and injury. Our data demonstrate that NK-1R is expressed in both aortic and vein-originated endothelial cells and that ox-LDL treatment induces NK-1R expression. Treatment with aprepitant suppresses induction of endothelial vascular adhesion molecule (VCAM-1 and E-selectin) and cytokine by ox-LDL. The presence of aprepitant mitigates adhesion of monocytes to endothelial cells and the reduction in eNOS/NO triggered by ox-LDL. Mechanistically, we demonstrate that aprepitant suppresses ERK5-KLF2 axis activation. Silencing of KLF2 abolishes the inhibitory role of aprepitant on ox-LDL-induced inflammatory response, suggesting that its action is dependent on KLF2. Collectively, our data support that aprepitant exerts an anti-inflammatory effect. Further research is required to investigate the therapeutic potential of aprepitant in vascular inflammation resulting from atherosclerosis.

Mild traumatic brain injury (mTBI) in a murine model increases survival to a bacterial pulmonary challenge compared with blunt tail trauma (TT). We hypothesize substance P and its receptor, the neurokinin 1 receptor (NK1R; official name TACR1), play a role in the increased survival of mTBI mice. Mice were subjected to mTBI or TT, and 48 hours after trauma, the levels of NK1R mRNA and protein were significantly up-regulated in mTBI lungs. Examination of the lung 48 hours after injury by microarray showed significant differences in the expression of 433 gene sets between groups, most notably genes related to intercellular proteins. Despite down-regulated gene expression of connective proteins, the presence of an intact pulmonary vasculature was supported by normal histology and bronchoalveolar lavage protein levels. To determine whether these mTBI-induced lung changes benefited in vivo responses, two chemotactic stimuli (a CXCL1 chemokine and a live Pseudomonas aeruginosa infection) were administered 48 hours after trauma. For both stimuli, mTBI mice recruited more neutrophils to the lung 4 hours after instillation (CXCL1: mTBI = 6.3 ± 1.3 versus TT = 3.3 ± 0.7 neutrophils/mL; Pseudomonas aeruginosa: mTBI = 9.4 ± 1.4 versus TT = 5.3 ± 1.1 neutrophils/mL). This study demonstrates that the downstream consequences of mTBI on lung NK1R levels and connective protein expression enhance neutrophil recruitment to a stimulus that may contribute to increased survival.

Chemoproteomic approaches to identify ligand-receptor interactions have gained popularity. However, identifying transmembrane receptors remains challenging. A new trifunctional probe to aid the nonbiased identification of such receptors was developed and synthesized using a convenient seven-step synthesis. This probe contained three functional groups: 1) an N-hydroxysuccinimide ester for ligand-coupling through free amines, 2) a diazirine moiety to capture the receptor of interest upon irradiation with UV light, and 3) a biotin group which allowed affinity purification of the final adduct using streptavidin. The interaction between the G protein–coupled tachykinin neurokinin 1 (NK1) receptor, expressed in an inducible manner, and the peptidic ligand substance P was used as a test system. Liquid chromatography–mass spectrometry analysis confirmed successful coupling of the probe to substance P, while inositol monophosphate accumulation assays demonstrated that coupling of the probe did not interfere substantially with the substance P–NK1 receptor interaction. Confocal microscopy and western blotting provided evidence of the formation of a covalent bond between the probe and the NK1 receptor upon UV activation. As proof of concept, the probe was used in full ligand-based receptor-capture experiments to identify the substance P–binding receptor via liquid chromatography–tandem mass spectrometry, resulting in the successful identification of only the NK1 receptor. This provides proof of concept toward general utilization of this probe to define interactions between ligands and previously unidentified plasma-membrane receptors.

Trichothecene mycotoxins, a family of common contaminants on cereal grains, are known to negatively impact human and animal health with adverse effect on food consumption being of particular concern. T-2 toxin has been previously demonstrated to induce anorectic response in several animal species including mouse, rat, rabbit. Although the T-2 toxin-induced anorectic response has been associated with the release of gut satiety hormone, much less is known about the role of neurotransmitter in this response. To address this gap, we employed a nocturnal mouse food refusal model to test the hypothesis that neurotransmitters 5-hydroxytryptamine (5-HT) and substance P (SP) mediate anorexia induction by T-2 toxin. Elevations of plasma 5-HT and SP markedly corresponded to anorexia induction following oral exposure to T-2 toxin. Direct administration of exogenous 5-HT and SP induced anorectic responses similar to T-2 toxin. The 5-HT3 receptor (5-HT3R) antagonist granisetron evoked a dose-dependent attenuation of both 5-HT- and T-2 toxin-induced anorectic responses. Pretreatment with neurokinin-1 receptor (NK-1R) antagonist Emend® dose-dependently attenuated both SP- and T-2 toxin-induced anorectic responses. To summarize, the results suggest that both 5-HT and SP play important roles in anorexia induction by T-2 toxin. 5-HT is more potent and long-acting than SP in this response.

The human immunodeficiency virus (HIV) envelope, via a key extracellular amino acid sequence, may simulate the functionality of native undecapeptide substance P (SP) acting through the host's neurokinin 1 (SP preferring) receptor (NK-1R). Human monocytes and macrophages express both NK-1Rs and SP. In HIV/AIDS the NK-1R may function as a chemokine-like G-protein coupled co-receptor that: 1) fuses to the outer envelope of HIV; 2) enables intracellular entry of the envelope-capsid-NK-1R complex; 3) co-opts immune defence via its physiological interaction with the SP-like envelope; 4) may contribute to resistance of CD4/chemokine entry inhibitor type drugs; 5) relaxes the blood-brain barrier to support entry of the HIV into the central nervous system, and 6) mediates most of the common clinical sequelae of HIV/AIDS (encephalopathy and AIDS dementia complex). The data support the idea that NK-1R antagonists could be useful to treat HIV/AIDS. Keywords: human immunodeficiency virus; NK-1 receptor; NK-1 receptor antagonist; aprepitant; fusion protein; virus.

Background Neurotransmitter substance P (SP) and its preferred neurokinin-1 receptor (NK1R) have been implicated in the treatment of affective and addiction disorders. Despite promising preclinical data on antidepressant action, the clinical trials of NK1R antagonists in major depression have been disappointing. There are no direct in vivo imaging studies on NK1R characteristics in patients with a major depressive disorder (MDD). Methods In this cross-sectional case-control study, we recruited nine never-medicated patients with moderate to severe MDD and nine matched healthy controls. NK1R availability (NK1R binding potential, BPND) was measured with in vivo 3-D positron emission tomography and a specific NK1 receptor tracer [18F]SPA-RQ. Clinical symptoms were assessed with the 17-item Hamilton Rating Scale for Depression (HAM-D17). Results NK1R-BPND did not differ statistically significantly between patients with MDD and healthy controls. HAM-D17 total scores (range 21–32) correlated positively with NK1R-BPND in cortical and limbic areas. HAM-D17 subscale score for anxiety symptoms correlated positively with NK1R-BPND in specific brain areas implicated in fear and anxiety. Limitations Small sample size. Low variability in the clinical HAM-D subscale ratings may affect the observed correlations. Conclusions Our preliminary results do not support a different baseline expression of NK1Rs in a representative sample of never-medicated patients with MDD during a current moderate/severe depressive episode. The modulatory effect of NK1Rs on affective symptoms is in line with early positive results on antidepressant action of NK1 antagonists. However, the effect is likely to be too weak for treatment of MDD with NK1R antagonists alone in clinical practice.

Background: Neuroblastoma is the commonest malignancy in neonates. Long non-coding RNA (lncRNA) RNA component of mitochondrial RNA processing endoribonuclease (RMRP) has been reported to be an oncogenic factor in some malignancies. However, its roles and molecular mechanisms in neuroblastoma progression are poor defined. Methods: The expression of RMRP, microRNA-206 (miR-206), and tachykinin-1 receptor (TACR1) mRNA was measured by RT-qPCR assay. Protein levels of TACR1, phosphorylated extracellular signal-regulated kinases (ERK) 1/2 (p-ERK1/2) and ERK1/2 were detected by western blot assay. Cell proliferation was assessed by CCK-8 and colony formation assays. Cell migratory and invasive capacities were determined using Transwell migration and invasion assays. The interaction between miR-206 and RMRP or TACR1 was verified by luciferase assay. The roles and molecular mechanisms of RMRP knockdown on the growth of neuroblastoma xenografts were examined in vivo. Results: RMRP was highly expressed in neuroblastoma tissues. RMRP knockdown inhibited proliferation, migration and invasion in neuroblastoma cells. Moreover, TACR1 was a target of miR-206 and RMRP performed as a molecular sponge of miR-206 to sequester miR-206 from TACR1 in neuroblastoma cells. TACR1 overexpression abrogated the inhibitory effect of RMRP downregulation on neuroblastoma cell progression by activating ERK1/2 pathway. Inhibition of TACR1 and ERK1/2 pathway abated RMRP-mediated pro-proliferation effect in neuroblastoma cells. RMRP knockdown hindered neuroblastoma xenograft growth by regulating miR-206/TACR1 axis via inactivating ERK1/2 pathway in vivo. Conclusion: RMRP knockdown hindered the tumorigenesis and progression of neuroblastoma by regulating miR-206/TACR1 axis via inactivating ERK1/2 pathway, hinting a potential therapeutic target for neuroblastoma.

Introduction The biological action of Substance P (SP) is mediated mainly by NK-1 receptors (NK1R) followed by NK2 receptors (NK2R). Aberrant expression of NK1R and NK2R has been identified in various carcinomas. The role of Substance P and its receptors, especially NK2R in cancer progression is not entirely known and there are conflicting results in the literature demonstrating the need for further investigation. In the current study, we examined the effects of SP and antagonists selective for the NK1R and NK2R in breast carcinoma cells metastasize to vital organs. Methods The effects of highly potent and selective non-peptide mouse NK1R and NK2R antagonists RP 67,580 and GR 159897, respectively, as well as SP and SP methyl ester, on both metastatic (4THM, 4TBM, 4TLM, 4T1) and non-metastatic (67NR) breast cancer cells were determined. Results NK1R and NK2R were over expressed in metastatic breast cells compared to non-metastatic cells. The NK1R antagonist at a 30 μM dose inhibited cell growth and induced cell death in metastatic cells while enhancing phosphorylation of Akt, the latter response not observed in the non-metastatic 67NR cells. Blocking the action of SP at the NK2R (30 μM antagonist) suppressed cellular proliferation in all the cell lines examined, with a response less prominent than that of the NK1R antagonist. Differently, the NK2R antagonist increased phosphorylation of p38 and enhanced MIP-2 secretion. SP and the SP methyl ester neither altered cell proliferation nor the effects of NK1R and NK2R antagonists in the metastatic cell lines. Conclusions Increased sensitivity of metastatic breast carcinoma cells to NK1R and NK2R antagonists suggest potential therapeutic value of antagonists in metastatic disease. NK1R and NK2R in metastatic breast carcinoma cells react differently to agonists and antagonists. These findings together with previously published data demonstrate that differential consequences of receptor antagonists and SP may inhibit breast cancer growth and metastasis.

Background Myocardial ischaemia/reperfusion (I/R) injury is a major cause of morbidity and mortality associated with coronary heart disease. During I/R injury, cardiomyocytes undergo cell death including apoptosis and necrosis with increased frequency, and this leads to compensatory cardiac hypertrophy, dysfunction, ventricular remodelling, and ultimately, heart failure. Accumulating evidence indicates that substance P is cardio-protective following I/R primarily due to its potent coronary vasodilator actions. However, its direct effect on cardiomyocytes in vitro remains controversial. Methods An hypoxia/reoxygenation (H/R)-induced cell death model was established to mimic I/R injury, in which the effect of substance P (SP) pretreatment on H9C2 cardiomyocytes and the mechanism of action were explored. Results Substance P was demonstrated to inhibit H/R-induced apoptosis. Phosphorylated-Akt (p-Akt) was decreased in H/R groups and was increased by SP pretreatment. Inhibition of p-Akt reduced the beneficial effects of SP in reducing apoptosis, whereas activation of Akt failed to provide additional improvement in the presence of SP. This suggests a key role for Akt in the process of reduced apoptosis by SP following H/R injury. In addition, the NK1-receptor antagonist prohibited the anti-apoptotic effect of SP. Conclusions This study indicates that SP pretreatment attenuates H/R-induced apoptosis via the Akt signalling pathway and the NK1-receptor.

Severe ocular surface diseases can lead to limbal stem cell deficiency (LSCD), which is accompanied by defective healing. We aimed to evaluate the role of the substance P (SP)/neurokinin-1 receptor (NK1R) pathway in corneal epithelium wound healing in a pre-clinical model of LSCD. SP ablation or NK1R blockade significantly increased epithelial wound healing (p 0.001) and corneal transparency (p 0.001), compared with wild type (WT). In addition, a reduced number of infiltrating goblet and conjunctival cells (p 0.05) and increased number of epithelial stem cells (p 0.01), which also expressed NK1R, was observed. The mammalian target of rapamycin (mTOR) pathway was significantly inhibited (p 0.05) and expression of γH2AX was significantly reduced (p 0.05) after SP ablation. These results suggest that excessive expression of SP is associated with LSCD and results in accelerated senescence and exhaustion of residual stem cells. Topical treatment with NK1R antagonist ameliorates clinical signs associated with LSCD and could be used as an adjuvant treatment in LSCD.

The intrathecal (i.t.) injection of substance P (SP) and N-methyl-D-aspartate (NMDA) induce transient nociceptive response by activating neurokinin (NK) 1 and NMDA receptors, respectively. We have recently reported that angiotensin (Ang) (1-7), an N-terminal fragment of Ang II, could alleviate several types of pain including neuropathic and inflammatory pain by activating spinal MAS1. Here, we investigated whether Ang (1-7) can inhibit the SP- and NMDA-induced nociceptive response. The nociceptive response induced by an i.t. injection of SP or NMDA was assessed by measuring the duration of hindlimb scratching directed toward the flank, biting and/or licking of the hindpaw or the tail for 5 min. Localization of MAS1 and either NK1 or NMDA receptors in the lumbar superficial dorsal horn was determined by immunohistochemical observation. The nociceptive response induced by SP and NMDA was attenuated by the i.t. co-administration of Ang (1-7) (0.03-3 pmol) in a dose-dependent manner. The inhibitory effects of Ang (1-7) (3 pmol) were attenuated by A779 (100 pmol), a MAS1 antagonist. Moreover, immunohistochemical analysis showed that spinal MAS1 co-localized with NK1 receptors and NMDA receptors on cells in the dorsal horn. Taken together, the i.t. injection of Ang (1-7) attenuated the nociceptive response induced by SP and NMDA via spinal MAS1, which co-localized with NK1 and NMDA receptors. Thus, the spinal Ang (1-7)/MAS1 pathway could represent a therapeutic target to effectively attenuate spinal pain transmission caused by the activation of NK1 or NMDA receptors.

Orientador: Alexandre Augusto Zaia Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba Resumo: Os cimentos endodônticos são agentes de união entre a guta percha e as paredes do canal radicular. Embora fiquem na maioria dos casos contidos no interior do canal, por vezes podem ser extruídos até o ligamento periodontal e pouco se sabe sobre o potencial destes materiais de causar alguma sensação dolorosa nestas condições. Dentre os mediadores químicos inflamatórios, a substância P se destaca por estar relacionada à fisiologia da dor. Assim, o objetivo deste estudo foi avaliar a citotoxicidade de quatro cimentos endodônticos e se eles são capazes de elevar a produção da substância P e de seus receptores NK1 em uma cultura de fibroblastos da linhagem MRC-5. Assim, a citotoxicidade de quatro cimentos endodônticos (AH Plus, Endométhasone N, Endoseal e MTA Fillapex) foi verificada pelo ensaio de citotoxicidade 3-(4,5-dimetiltiazol-2-il)-2,5-difenil brometo de tetrazólio (MTT) para a aquisição da porcentagem de viabilidade celular em cada uma das diluições. As células foram então expostas aos extratos dos materiais testados em duas diluições escolhidas (1:4 e 1:8) e, após 24 horas, os sobrenadantes foram coletados para a realização do ensaio de imunoabsorção enzimática (Elisa) para substância P. Já as células tiveram o seu RNA extraído para a realização de PCR em tempo real, que quantificou a expressão dos genes TAC1 e TACR1 referente à substância P e receptor NK1, respectivamente. Observou-se que o cimento MTA Fillapex foi o mais citotóxico dentre os materiais testados e este cimento endodôntico foi o único material capaz de induzir a produção de substância P após o contato de seu extrato com a cultura celular. Além disso, todas as amostras expostas aos extratos dos cimentos endodônticos apresentaram expressão dos genes TAC1 e TACR1 aumentada, quando comparados ao grupo controle negativo. Portanto, de acordo com os resultados do presente estudo, conclui-se que todos os cimentos endodônticos são citotóxicos e que a expressão de substância P foi influenciada pelo contato da cultura de células com a elução do cimento endodôntico MTA Fillapex. Contudo, houve um aumento da expressão dos genes codificantes da substância P e do seu receptor NK1 em todas as amostras Abstract: Root canal sealers are bond agents between gutta percha and the root canal walls. Although they are in most cases contained within the canal, they are able to be extruded into the periodontal ligament sometimes, and there isn¿t many knowledgement about the potential of these materials to cause any painful sensation under these conditions. Among the chemical mediators of inflammation, substance P stands out because it is directly related to the physiology of pain. Thus, the aim of the present study was to analyze the potential of substance P and NK1 receptors production by different endodontic root canal sealers, and their cytotoxicity, when in contact with MRC5 fibroblasts cell culture. So, the cytotoxicity of four endodontic root canal sealers (AH Plus, Endométhasone N, Endoseal and MTA Fillapex) were verified by the cell viability test 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to gain the percentage of viable cells in each of the dilutions. The cells were then exposed to the extracts of the materials in two chosen dilutions (1:4 and 1:8). After 24 hours the supernatants were collected for the enzyme-linked immunosorbent assay (ELISA) for substance P, and the cell cultures had their RNA extracted for real-time PCR, which quantified the expression of TAC1 and TACR1 genes which encodes substance P and NK1 receptors, respectively. The results showed that MTA Fillapex was the most cytotoxic materials tested. In addition, it was the only material able to induce the production of substance P by fibroblasts. All samples showed increased TAC1 and TACR1 gene expression when compared to the negative control group. Therefore, we can conclude that all endodontic root canal sealer materials are cytotoxic, but the expression of substance P was induced only by MTA Fillapex. However, there was an increased expression of the genes TAC1 and TACR1 influenced by all root canal sealers Mestrado Endodontia Mestra em Clínica Odontológica CNPQ 132882/2015-9

Anxiety disorder is a psychiatric disorder characterized by extreme fear or worry. It is highly prevalent worldwide which affects daily life and is also an enormous health burden. Neurokinin 1 receptor (NK1R) is a G protein coupled receptor, expressed in both central and peripheral nervous system, involved in affective behaviors. NK1R has established role in anxiety and it is also an important target for pathogenesis of anxiety disorder. Therefore, it has been hypothesized in previous studies that the blockades of NK1R may have antidepressant and anxiolytic effects. The present study deals with the molecular mechanism of protective activity of eugenol against anxiolytic disorder. A pre-clinical animal study was performed on 42 BALB/c mice. Animals were given stress through conventional restrain model. The mRNA expression of NK1R was analyzed by real time RT-PCR. Moreover, the NK1R protein expression was also examined by immunohistochemistry in whole brain and mean density was calculated. The mRNA and protein expressions were found to be increased in animals given anxiety as compared to the normal control. Whereas, the expressions were decreased in the animals treated with eugenol and its liposome-based nanocarriers in a dose dependent manner. However, the results were better in animals treated with nanocarriers as compared to the compound alone. It is concluded that the eugenol and its liposome-based nanocarriers exert anxiolytic activity by down-regulating NK1R protein expression in mice.