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BACKGROUND The mechanisms underlying mucosal eosinophilia in chronic rhinosinusitis with nasal polyps (CRSwNP) remain poorly clarified. The nervous system and neuropeptides play an important role in the regulation of immune response. Herein we explore the expression and function of hemokinin-1 (HK-1), a newly identified tachykinin, along with its receptor neurokinin 1 receptor (NK1R) in CRSwNP. METHODS HK-1, NK1R, and C-C motif chemokine ligand 24 (CCL24) expression in nasal tissues (53 eosinophilic CRSwNP, 32 non-eosinophilic CRSwNP, and 33 controls) was investigated by quantitative reverse transcript polymerase chain reaction (RT-PCR) and immunofluorescence staining. THP-1, a human monocytic leukemia cell line, and eosinophilic polyp tissues were stimulated with HK-1. Cells, tissues, and culture supernatants were subsequently collected for detection of the production of various inflammatory cytokines and chemokines by quantitative RT-PCR and enzyme-linked immunoassay. RESULTS HK-1 and NK1R mRNA and protein expression were upregulated in eosinophilic and non-eosinophilic nasal polyps compared with control tissues, with eosinophilic polyps demonstrating a higher upregulation compared with that of non-eosinophilic polyps. Eosinophils constituted the major source of HK-1, whereas macrophages were the predominant cell type exhibiting NK1R in eosinophilic polyps. HK-1 induced CCL24 production from macrophages differentiated from THP-1 cells; this was abolished by an NK1R antagonist. HK-1 also induced CCL24 production from ex vivo-cultured eosinophilic nasal polyps. CCL24 was expressed by macrophages in eosinophilic but not non-eosinophilic polyps. The expression level of HK-1 correlated with CCL24 expression and tissue eosinophilia in eosinophilic nasal polyps. CONCLUSION Eosinophil-derived HK-1 induces CCL24 production from macrophages and therefore exaggerates eosinophilic inflammation in CRSwNP.

Cystathionine-&gamma, lyase (CSE) isa hydrogen sulfide (H2S)-synthesizing enzyme that promotesinflammation by upregulating H2S in sepsis. Liver sinusoidal endothelial cells (LSECs) are fenestrated endothelial cells (liver sieve) that undergo alteration during sepsis and H2S plays a role in this process. Substance P (SP) is encoded by the preprotachykinin A (PPTA) gene, and promotes inflammation in sepsis, however, its regulation by H2S is poorly understood. Furthermore, the interaction between H2S and SP in modulating LSEC fenestrations following sepsis remains unclear. This study aimed to investigate whether CSE/H2S regulates SP and the neurokinin-1 receptor (NK-1R) andmodulates fenestrations in LSECs following caecalligation and puncture (CLP)-induced sepsis. Here we report thatthe absence of either CSE or H2S protects against liver sieve defenestration and gaps formation in LSECsin sepsis by decreased SP-NK-1R signaling. Following sepsis, there is an increased expression of liver CSE and H2S synthesis, and plasma H2S levels, which were aligned with higher SP levels in the liver, lungs and plasma and NK-1R in the liver and lungs. The genetic deletion of CSE led to decreased sepsis-induced SP and NK-1R in the liver, lungs and plasma SP suggesting H2S synthesized through CSE regulates the SP-NK-1R pathway in sepsis. Further, mice deficient in the SP-encoding gene (PPTA) preservedsepsis-induced LSEC defenestrationand gaps formation, as seen by maintenance of patent fenestrations and fewer gaps. In conclusion, CSE/H2S regulates SP-NK-1R and modulates LSEC fenestrations in sepsis.

Background Substance P (SP) plays an important role in several types of cancer promotion and progression by binding to its preferential neurokinin 1 receptor (NK1R). However, the clinical significance and downstream mechanism of NK1R in esophageal squamous cell carcinoma (ESCC) have not been elucidated. The aim of this study was to investigate the role of SP/NK1R in the proliferation of ESCC and to screen related downstream molecules. Methods In the current investigation, the expression of NK1R was detected via immunohistochemistry (IHC), western blot (WB) analysis and real-time reverse transcription-polymerase chain reaction (RT-qPCR) in ESCC tissues and cell lines. Thereafter, the optimal concentration of SP was determined in vitro. The proliferation ability of SP/NK1R was assessed by cell counting kit-8 (CCK-8) and colony formation assays and subcutaneous tumour formation in nude mice with EC109 cells. Moreover, the related downstream molecules were screened by performing isobaric tags for relative and absolute quantitation (iTRAQ) protein spectrum analysis. Results NK1R was upregulated in ESCC, and its overexpression correlated with larger tumour size, deeper tumour invasion, more perineural invasion and eventually caused poorer overall survival (OS). Both intrinsic and SP-activated NK1R upregulation could promote the proliferation and clonogenic capacity of ESCC cells. In nude mice, tumour growth was suppressed by EC109 cells of NK1R downregulation. Further experiments demonstrated that Hairy and Enhancer of Split 1 (Hes1) was markedly reduced upon NK1R downregulation in EC109 cell lines and could regulate cell proliferation in the downstream of SP/NK1R. Conclusions The significant role of NK1R in mediating ESCC cell proliferation depended on the activation of SP and might be related to the downstream regulation of Hes1.

Visual Abstract, Reelin (Reln) and Disabled-1 (Dab1) participate in the Reln-signaling pathway and when either is deleted, mutant mice have the same spinally mediated behavioral abnormalities, increased sensitivity to noxious heat and a profound loss in mechanical sensitivity. Both Reln and Dab1 are highly expressed in dorsal horn areas that receive and convey nociceptive information, Laminae I–II, lateral Lamina V, and the lateral spinal nucleus (LSN). Lamina I contains both projection neurons and interneurons that express Neurokinin-1 receptors (NK1Rs) and they transmit information about noxious heat both within the dorsal horn and to the brain. Here, we ask whether the increased heat nociception in Reln and dab1 mutants is due to incorrectly positioned dorsal horn neurons that express NK1Rs. We found more NK1R-expressing neurons in Reln-/- and dab1-/- Laminae I–II than in their respective wild-type mice, and some NK1R neurons co-expressed Dab1 and the transcription factor Lmx1b, confirming their excitatory phenotype. Importantly, heat stimulation in dab1-/-mice induced Fos in incorrectly positioned NK1R neurons in Laminae I–II. Next, we asked whether these ectopically placed and noxious-heat responsive NK1R neurons participated in pain behavior. Ablation of the superficial NK1Rs with an intrathecal injection of a substance P analog conjugated to the toxin saporin (SSP-SAP) eliminated the thermal hypersensitivity of dab1-/- mice, without altering their mechanical insensitivity. These results suggest that ectopically positioned NK1R-expressing neurons underlie the heat hyperalgesia of Reelin-signaling pathway mutants, but do not contribute to their profound mechanical insensitivity.

Tachykinins (TKs) include an evolutionarily conserved group of small bio-active peptides which possess a common carboxyl-terminal sequence, Phe-X-Gly-Leu-Met-NH2. TKs also have been shown to have implications in different steps of carcinogenesis, such as angiogenesis, mitogenesis, metastasis, and other growth-related events. The biological actions of substance P (SP), as the most important member of the TK family, are mainly mediated through a G protein-coupled receptor named neurokinin-1 receptor (NK1R). More recently, it has become clear that SP/NK1R system is involved in the initiation and activation of signaling pathways involved in cancer development and progression. Therefore, SP may contribute to triggering a variety of effector mechanisms including protein synthesis and a number of transcription factors that modulate the expression of genes involved in these processes. The overwhelming insights into the blockage of NK1R using specific antagonists could suggest a therapeutic approach in cancer therapy. In this review, we focus on evidence supporting an association between the signaling pathways of the SP/NK1R system and cancer cell proliferation and development.

Context: Atopic dermatitis (AD) is a chronic, inflammatory, itching skin disorder, which may worsen due to stress, depression and anxiety. Tachykinins may be involved in inflammation signaling as well as they may have a role in stress, depression and anxiety. Objective: This study aimed to measure the expression of tachykinin markers, in the skin of patients with AD, and the correlation of these tachykinins with clinical and psychodemographic parameters. Materials and methods: Twenty-eight adult patients with AD were investigated regarding tachykinin expression in skin biopsies, using an immunohistochemical technique. The patients were characterized with clinical and psychodemographic parameters. Results: The number of substance P and neurokinin (NK)A positive nerve fibers, as well as NKA positive mononuclear dermal cells, was increased in lesional compared to non-lesional skin. Interestingly, the depression score and the number of dermal NK-1 receptor (R) positive cells in lesional as well as in non-lesional skin showed a correlation. Conclusion: These findings indicate an upregulation of the tachykinergic system in the inflamed skin of AD.

The substance P/neurokinin-1 receptor system has been implicated in tumor cell proliferation. Neurokinin-1 receptor has been identified in different solid tumors but not frequently in hematopoietic malignant cells. We investigated the presence of the Neurokinin-1 receptor in acute myeloid leukemia cell lines (KG-1 and HL-60), demonstrating that acute myeloid leukemia cell lines overexpress the truncated Neurokinin-1 receptor isoform compared with lymphocytes from healthy donors. Using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) method, we demonstrated that substance P induced cell proliferation in both acute myeloid leukemia cell lines. We also observed that four different Neurokinin-1 receptor antagonists (L-733,060, L-732,138, CP 96–345 and aprepitant) elicited inhibition of acute myeloid leukemia cell growth lines in a concentration-dependent manner, while growth inhibition was only marginal in lymphocytes; the specific antitumor action of Neurokinin-1 receptor antagonists occurs via the Neurokinin-1 receptor, and leukemia cell death is due to apoptosis. Finally, administration of high doses of daily intraperitoneal fosaprepitant to NOD scid gamma mice previously xenografted with the HL60 cell line increased the median survival from 4 days (control group) to 7 days (treated group) (p = 0.059). Taken together, these findings suggest that Neurokinin-1 receptor antagonists suppress leukemic cell growth and may be considered to be potential antitumor drugs for the treatment of human acute myeloid leukemia.

Kidney ischemia, characterized by insufficient supply of oxygen and nutrients to renal epithelial cells, is the main cause of acute kidney injury and an important contributor to mortality world-wide. Earlier research implicated a G-protein coupled receptor (NK1R) in the death of kidney epithelial cells in ischemia-like conditions. P21-associated kinase 1 (PAK1) is involved in signalling by several G-proteins. We explored the consequences of PAK1 inhibition for cell survival under the conditions of reduced glucose and oxygen. Inhibition of PAK1 by RNA interference, expression of a dominant-negative mutant or treatment with small molecule inhibitors greatly reduced the death of cultured kidney epithelial cells. Similar protection was achieved by treating the cells with inhibitors of MEK1, in agreement with the prior reports on PAK1-MEK1 connection. Concomitant inhibition of NK1R and PAK1 offered no better protection than inhibition of NK1R alone, consistent with the two proteins being members of the same pathway. Furthermore, NK1R, PAK and MEK inhibitors reduced the induction of TRAIL in ischemia-like conditions. Considering the emerging role of TRAIL in ischemia-mediated cell death, this phenomenon may contribute to the protective effects of these small molecules. Our findings support further exploration of PAK and MEK inhibitors as possible agents to avert ischemic kidney injury.

Endometriotic lesions are known to be hyperinnervated, especially in lesions of deep endometriosis (DE), which are frequently in close proximity to various nerve plexuses. DE lesions typically have higher fibromuscular content than that of ovarian endometriomas (OE) lesions, but the underlying reason remains elusive. Aside from their traditional role of pain transduction, however, whether or not sensory nerves play any role in the development of endometriosis is unclear. Here, we show that, thorough their respective receptors neurokinin receptor 1 (NK1R), calcitonin receptor like receptor (CRLR), and receptor activity modifying protein 1 (RAMP-1), neuropeptides substance P (SP) and calcitonin gene related peptide (CGRP) induce epithelial-mesenchymal transition (EMT), fibroblast-to-myofibroblast transdifferentiation (FMT) and further turn stromal cells into smooth muscle cells (SMCs) in endometriotic lesions, resulting ultimately in fibrosis. We show that SP and CGRP, or the rat dorsal root ganglia (DRG) supernatant, through the induction of NK1R and CGRP/CRLR/RAMP-1 signaling pathways, promoted EMT, FMT and SMM in endometriosis, resulting in increased migratory and invasive propensity, cell contractility, production of collagen, and eventually to fibrosis. Neutralization of NK1R and/or CGRP/CRLR/RAMP-1 abrogated these processes. Extended exposure of endometriotic stromal cells to SP and/or CGRP or the DRG supernatant induced increased expression of α-SMA, desmin, oxytocin receptor, and smooth muscle myosin heavy-chain. Finally, we show that DE lesions had significantly higher nerve fiber density, increased staining levels of α-SMA, NK1R, CRLR, and RAMP-1, concomitant with higher lesional fibrotic content than that of OE lesions. The extent of lesional fibrosis correlated positively with the staining levels of NK1R, CRLR, and RAMP-1, as well as the nerve fiber density in lesions. Thus, this study provides another piece of evidence that sensory nerves play an important role in promoting the development and fibrogenesis of endometriosis. It explains as why DE frequently have higher fibromuscular content than that of OE, highlights the importance of lesional microenvironment in shaping the lesional fate, gives more credence to the idea that ectopic endometrium is fundamentally wounds that go through repeated tissue injury and repair, and should shed much needed light into the pathophysiology of endometriosis.

The highly pathogenic H5N1 (HK483) viral infection causes a depressed hypercapnic ventilatory response (dHCVR, 20%↓) at 2 days postinfection (dpi) and death at 7 dpi in mice, but the relevant mechanisms are not fully understood. Glomus cells in the carotid body and catecholaminergic neurons in locus coeruleus (LC), neurokinin 1 receptor (NK1R)-expressing neurons in the retrotrapezoid nucleus (RTN), and serotonergic neurons in the raphe are chemosensitive and responsible for HCVR. We asked whether the dHCVR became worse over the infection period with viral replication in these cells/neurons. Mice intranasally inoculated with saline or the HK483 virus were exposed to hypercapnia for 5 min at 0, 2, 4, or 6 dpi, followed by immunohistochemistry to determine the expression of nucleoprotein of H5N1 influenza A (NP) alone and coupled with 1) tyrosine hydroxylase (TH) in the carotid body and LC, 2) NK1R in the RTN, and 3) tryptophan hydroxylase (TPH) in the raphe. HK483 viral infection blunted HCVR by ∼20, 50, and 65% at 2, 4, and 6 dpi. The NP was observed in the pontomedullary respiratory-related nuclei (but not in the carotid body) at 4 and 6 dpi, especially in 20% of RTN NK1R, 35% of LC TH, and ∼10% raphe TPH neurons. The infection significantly reduced the local NK1R or TPH immunoreactivity and population of neurons expressing NK1R or TPH. We conclude that the HK483 virus infects the pontomedullary respiratory nuclei, particularly chemosensitive neurons in the RTN, LC, and raphe, contributing to the severe depression of HCVR and respiratory failure at 6 dpi.

Neurokinins (or tachykinins) are peptides that modulate a wide variety of human physiology through the neurokinin G protein-coupled receptor family, implicated in a diverse array of pathological processes. Here we report high-resolution crystal structures of the human NK1 receptor (NK1R) bound to two small-molecule antagonist therapeutics – aprepitant and netupitant and the progenitor antagonist CP-99,994. The structures reveal the detailed interactions between clinically approved antagonists and NK1R, which induce a distinct receptor conformation resulting in an interhelical hydrogen-bond network that cross-links the extracellular ends of helices V and VI. Furthermore, the high-resolution details of NK1R bound to netupitant establish a structural rationale for the lack of basal activity in NK1R. Taken together, these co-structures provide a comprehensive structural basis of NK1R antagonism and will facilitate the design of new therapeutics targeting the neurokinin receptor family., Neurokinin receptors are G protein-coupled receptors. Here the authors present three crystal structures of the neurokinin 1 receptor (NK1R) in complex with small-molecule antagonists including aprepitant and netupitant and observe that these clinically approved compounds induce a conformational change in the receptor.

Morphine, which acts through opioid receptors, is one of the most efficient analgesics for the alleviation of severe pain. However, its usefulness is limited by serious side effects, including analgesic tolerance, constipation, and dependence liability. The growing awareness that multifunctional ligands which simultaneously activate two or more targets may produce a more desirable drug profile than selectively targeted compounds has created an opportunity for a new approach to developing more effective medications. Here, in order to better understand the role of the neurokinin system in opioid-induced antinociception, we report the synthesis, structure&ndash, activity relationship, and pharmacological characterization of a series of hybrids combining opioid pharmacophores with either substance P (SP) fragments or neurokinin receptor (NK1) antagonist fragments. On the bases of the in vitro biological activities of the hybrids, two analogs, opioid agonist/NK1 antagonist Tyr-[d-Lys-Phe-Phe-Asp]-Asn-d-Trp-Phe-d-Trp-Leu-Nle-NH2 (2) and opioid agonist/NK1 agonist Tyr-[d-Lys-Phe-Phe-Asp]-Gln-Phe-Phe-Gly-Leu-Met-NH2 (4), were selected for in vivo tests. In the writhing test, both hybrids showed significant an antinociceptive effect in mice, while neither of them triggered the development of tolerance, nor did they produce constipation. No statistically significant differences in in vivo activity profiles were observed between opioid/NK1 agonist and opioid/NK1 antagonist hybrids.

International audience; We have proposed that cortical nNOS/NK1R interneurons have a role in sleep homeostasis. The hypocretins (orexins) are wake-promoting neuropeptides and hypocretin/orexin (Hcrt) neurons project to the cortex. Hcrt peptides affect deep layer cortical neurons, and Hcrt receptor 1 (Hcrtr1; Ox1r) mRNA is expressed in cortical nNOS/NK1R cells. Therefore, we investigated whether Hcrt neuron stimulation affects cingulate cortex nNOS/NK1R neurons. Bath application of HCRT1/orexin-A evoked an inward current and membrane depolarization in most nNOS/NK1R cells which persisted in tetrodotoxin; optogenetic stimulation of Hcrt terminals expressing channelrhodopsin-2 confirmed these results, and pharmacological studies determined that HCRTR1 mediated these responses. Single-cell RT-PCR found Hcrtr1 mRNA in 31% of nNOS/NK1R cells without any Hcrtr2 mRNA expression; immunohistochemical studies of Hcrtr1-EGFP mice confirmed that a minority of nNOS/NK1R cells express HCRTR1. When Hcrt neurons degenerated in orexin-tTA;TetO DTA mice, the increased EEG delta power during NREM sleep produced in response to 4 h sleep deprivation and c-FOS expression in cortical nNOS/NK1R cells during recovery sleep were indistinguishable from that of controls. We conclude that Hcrt excitatory input to these deep layer cells is mediated through HCRTR1 but is unlikely to be involved in the putative role of cortical nNOS/NK1R neurons in sleep homeostasis.

The NK(1) tachykinin G-protein–coupled receptor (GPCR) binds substance P, the first neuropeptide to be discovered in mammals. Through activation of NK(1)R, substance P modulates a wide variety of physiological and disease processes including nociception, inflammation, and depression. Human NK(1)R (hNK(1)R) modulators have shown promise in clinical trials for migraine, depression, and emesis. However, the only currently approved drugs targeting hNK(1)R are inhibitors for chemotherapy-induced nausea and vomiting (CINV). To better understand the molecular basis of ligand recognition and selectivity, we solved the crystal structure of hNK(1)R bound to the inhibitor L760735, a close analog of the drug aprepitant. Our crystal structure reveals the basis for antagonist interaction in the deep and narrow orthosteric pocket of the receptor. We used our structure as a template for computational docking and molecular-dynamics simulations to dissect the energetic importance of binding pocket interactions and model the binding of aprepitant. The structure of hNK(1)R is a valuable tool in the further development of tachykinin receptor modulators for multiple clinical applications.

Neurokinin-1 receptor (NK1R) signaling can be immunomodulatory and it can lead to preferential transmigration of CD14+CD16+ monocytes across the blood brain barrier, potentially promoting the development of inflammatory neurological diseases, such as neuroHIV. To evaluate how NK1R signaling alters monocyte biology, RNA sequencing was used to define NK1R-mediated transcriptional changes in different monocyte subsets. The data show that NK1R activation induces a greater number of changes in CD14+CD16+ monocytes (152 differentially expressed genes), than in CD14+CD16- monocytes (36 genes), including increases in the expression of NF-κB and components of the NLRP3 inflammasome pathway. These results suggest that NK1R may alter the inflammatory state of CD14+CD16+ monocytes, influencing the development of neuroinflammation.

The neurokinin-1 receptor plays a profound role in inflammatory processes and is involved in immune cell differentiation, cytokine release, and mast cell activation. Due to their similar peptide structures, the neurokinin-1 receptor does not discriminate between the endogenous ligands substance P (SP) and human hemokinin-1 (hHK-1), which both demonstrate biological receptor affinity. In addition, due to cross-reactivity, the current bioanalytical method of choice-immunoassays-also displays limitations in differentiating between these peptides. Thus, a recently developed mass spectrometric assay was utilized for the selective quantification of SP and hHK-1 in various biofluids and tissue. By applying the sample processing protocols developed, SP was quantified in porcine brain tissue (4.49 ± 0.53 nM), human saliva (113.3 ± 67.0 pM), and human seminal fluid (0.52 ± 0.15 nM) by mass spectrometric analysis. As previously reported, neither SP nor hHK-1 could be detected in human plasma by mass spectrometry. Comparison with analysis using a commercial immunoassay of the same plasma sample revealed SP like-immunoreactivity concentrations of 37.1-178.0 pM. The previously reported carboxylic acid of SP, whose identity was confirmed by high-resolution mass spectrometric analysis, did not show cross-reactivity in the applied immunoassay and did not contribute to SP-like immunoreactivity results. Subsequent compound discovery of the immunocaptured substance indicated the presence of a precursor of SP as possible cross-reactor in human plasma samples. The found cross-reactivity might be the cause for the high variance of SP plasma levels in former determinations.

Otilonium bromide (OB) is a spasmolytic drug successfully used for the treatment of irritable bowel syndrome (IBS). Its efficacy has been attributed to the block of L‐ and T‐type Ca2+ channels and muscarinic and tachykinin receptors in the smooth muscle. Furthermore, in healthy rats, repeated OB administration modified neurotransmitter expression and function suggesting other mechanisms of action. On this basis, we investigated whether repeated OB treatment prevented the functional and neurochemical changes observed in the colon of rats underwent to wrap restrain stress (WRS) a psychosocial stressor considered suitable to reproduce the main IBS signs and symptoms. In control, WRS and OB/WRS rats functional parameters were measured in vivo and morphological investigations were done ex vivo in the colon. The results showed that OB counteracts most of the neurotransmitters changes caused by WRS. In particular, the drug prevents the decrease in SP‐, NK1r‐, nNOS‐, VIP‐, and S100β‐immunoreactivity (IR) and the increase in CGRP‐, and CRF1r‐IR. On the contrary, OB does not affect the increase in CRF2r‐IR neurons observed in WRS rats and does not interfere with the mild mucosal inflammation due to WRS. Finally, OB per se increases the Mr2 expression in the muscle wall and decreases the number of the myenteric ChAT‐IR neurons. Functional findings show a significantly reduction in the number of spontaneous abdominal contraction in OB treated rats. The ability of OB to block L‐type Ca2+ channels, also expressed by enteric neurons, might represent a possible mechanism through which OB exerts its actions.

Substance P neuropeptide and its receptor, neurokinin-1 receptor (NK1R), are reported to present on the ocular surface. In this study, mice lacking functional NK1R exhibited an excessive desquamation of apical corneal epithelial cells in association with an increased epithelial cell proliferation and increased epithelial cell density, but decreased epithelial cell size. The lack of NK1R also resulted in decreased density of corneal nerves, corneal epithelial dendritic cells (DCs), and a reduced volume of basal tears. Interestingly, massive accumulation of CD11c+CD11b+ conventional DCs was noted in the bulbar conjunctiva and near the limbal area of corneas from NK1R−/− mice. After ocular HSV-1 infection, the number of conventional DCs and neutrophils infiltrating the infected corneas was significantly higher in NK1R−/− than C57BL/6J mice. This was associated with an increased viral load in infected corneas of NK1R−/− mice. As a result, the number of IFN-γ–secreting virus-specific CD4 T cells in the draining lymph nodes of NK1R−/− mice was much higher than in infected C57BL/6J mice. An increased number of CD4 T cells and mature neutrophils (CD11b+Ly6ghigh) in the inflamed corneas of NK1R−/− mice was associated with an early development of severe herpes stromal keratitis. Collectively, our results show that the altered corneal biology of uninfected NK1R−/− mice along with an enhanced immunological response after ocular HSV-1 infection causes an early development of herpes stromal keratitis in NK1R−/− mice.

Combination studies of neurokinin 1 (NK1) receptor antagonists and serotonin-selective reuptake inhibitors (SSRIs) have shown promise in preclinical models of depression. Such a combination may offer important advantages over the current standard of care. Herein we describe the discovery and optimization of an indazole-based chemotype to provide a series of potent dual NK1 receptor antagonists/serotonin transporter (SERT) inhibitors to overcome issues of ion channel blockade. This effort culminated in the identification of compound 9, an analogue that demonstrated favorable oral bioavailability, excellent brain uptake, and robust in vivo efficacy in a validated depression model. Over the course of this work, a novel heterocycle-directed asymmetric hydrogenation was developed to facilitate installation of the key stereogenic center.

There is now general agreement that neurokinin B (NKB) acts via neurokinin-3-receptor (NK3R) to stimulate secretion of GnRH and LH in several species, including rats, mice, sheep, and humans. However, the roles of two other tachykinins, substance P (SP) and neurokinin A, which act primarily via NK1R and NK2R, respectively, are less clear. In rodents, these signaling pathways can stimulate LH release and substitute for NKB signaling; in humans, SP is colocalized with kisspeptin and NKB in the mediobasal hypothalamus. In this study, we examined the possible role of these tachykinins in control of the reproductive axis in sheep. Immunohistochemistry was used to describe the expression of SP and NK1R in the ovine diencephalon and determine whether these proteins are colocalized in kisspeptin or GnRH neurons. SP-containing cell bodies were largely confined to the arcuate nucleus, but NK1R-immunoreactivity was more widespread. However, there was very low coexpression of SP or NK1R in kisspeptin cells and none in GnRH neurons. We next determined the minimal effective dose of these three tachykinins that would stimulate LH secretion when administered into the third ventricle of ovary-intact anestrous sheep. A much lower dose of NKB (0.2 nmol) than of neurokinin A (2 nmol) or SP (10 nmol) consistently stimulated LH secretion. Moreover, the relative potency of these three neuropeptides parallels the relative selectivity of NK3R. Based on these anatomical and pharmacological data, we conclude that NKB-NK3R signaling is the primary pathway for the control of GnRH secretion by tachykinins in ewes.

Ligand-receptor binding kinetics (i.e. association and dissociation rates) are emerging as important parameters for drug efficacy in vivo. Awareness of the kinetic behavior of endogenous ligands is pivotal, as drugs often have to compete with those. The binding kinetics of neurokinin 1 (NK1) receptor antagonists have been widely investigated while binding kinetics of endogenous tachykinins have hardly been reported, if at all. Therefore, the aim of this research was to investigate the binding kinetics of endogenous tachykinins and derivatives thereof and their role in the activation of the NK1 receptor. We determined the binding kinetics of seven tachykinins targeting the NK1 receptor. Dissociation rate constants (koff) ranged from 0.026±0.0029min-1 (Sar9,Met(O2)11-SP) to 0.21±0.015min-1 (septide). Association rate constants (kon) were more diverse: substance P (SP) associated the fastest with a kon value of 0.24±0.046nM-1min-1 while neurokinin A (NKA) had the slowest association rate constant of 0.001±0.0002nM-1min-1. Kinetic binding parameters were highly correlated with potency and maximal response values determined in label-free impedance-based experiments on U-251 MG cells. Our research demonstrates large variations in binding kinetics of tachykinins which correlate to receptor activation. These findings provide new insights into the ligand-receptor interactions of tachykinins and underline the importance of measuring binding kinetics of both drug candidates and competing endogenous ligands.

The neurokinin-1 receptor (NK1R) is implicated in the growth and metastasis of many tumors, including cancers of the brain (e.g., gliomas, glioblastomas, and astrocytomas), skin (e.g., melanomas), and neuroendocrine tissues (cancers of the breast, stomach, pancreas, larynx, and colon). Because overexpression of NK1R has been reported in most of these malignancies, we have undertaken designing an NK1R-targeted near-infrared (NIR) fluorescent dye for fluorescence-guided surgeries of these cancers. We demonstrate here that an NK1R-binding ligand linked to the NIR dye LS288 selectively accumulates in NK1R-expressing tumor xenografts with high affinity (Kd = 13 nM), allowing intraoperative imaging of these cancers in live mice. Because tumor accumulation is nearly quantitatively blocked by excess unlabeled ligand, and because NK1R-negative tumors and normal tissues display virtually no uptake, we conclude that the observed tumor retention is NK1R-mediated. Results on the synthesis, in vitro characterization, and animal testing of NK1R-targeted NIR dye are presented.

Receptors and their regulatory peptides are aberrantly expressed in tumors, suggesting a potential tumor therapy target. Human hemokinin-1 (hHK-1) is a tachykinin peptide ligand of the neurokinin-1 (NK1) receptor which is overexpressed in melanoma and other tumor tissues. Here, we investigated the role of hHK-1 and the NK1 receptor in melanoma cell migration. NK1 receptor expression was associated with melanoma metastatic potential. Treatment with hHK-1 significantly enhanced A375 and B16F10 melanoma cell migration and an NK1 receptor antagonist L732138 blocked this effect. MMP-2 and MT1-MMP expression were up-regulated in hHK-1-treated melanoma cells and cell signaling data suggested that hHK-1 induced phosphorylation of ERK1/2, JNK and p38 by way of PKC or PKA. Kinase activation led to increased MMP-2 and MT1-MMP expression and melanoma cell migration induced by hHK-1. Thus, hHK-1 and the NK1 receptor are critical to melanoma cell migration and each may be a promising chemotherapeutic target.

It is supposed that TRPA1 receptor can be activated by hydrogen sulphide (H2S). Here, we have investigated the role of TRPA1 receptor in H2S-induced [Ca(2+)]i increase in trigeminal ganglia (TRG) neurons, and the involvement of capsaicin-sensitive sensory nerves in H2S-evoked cutaneous vasodilatation. [Ca(2+)]i was measured with ratiometric technique on TRG neurons of TRPA1(+/+) and TRPA1(-/-) mice after NaHS, Na2S, allylisothiocyanate (AITC) or KCl treatment. Microcirculatory changes in the ear were detected by laser Doppler imaging in response to topical NaHS, AITC, NaOH, NaSO3 or NaCl. Mice were either treated with resiniferatoxin (RTX), or CGRP antagonist BIBN4096, or NK1 receptor antagonist CP99994, or K(+) ATP channel blocker glibenclamide. Alpha-CGRP(-/-) and NK1 (-/-) mice were also investigated. NaHS and Na2S increased [Ca(2+)]i in TRG neurons derived from TRPA(+/+) but not from TRPA1(-/-) mice. NaHS increased cutaneous blood flow, while NaOH, NaSO3 and NaCl did not cause significant changes. NaHS-induced vasodilatation was reduced in RTX-treated animals, as well as by pre-treatment with BIBN4096 or CP99994 alone or in combination. NaHS-induced vasodilatation was significantly smaller in alpha-CGRP(-/-) or NK1 (-/-) mice compared to wild-types. H2S activates capsaicin-sensitive sensory nerves through TRPA1 receptors and the resultant vasodilatation is mediated by the release of vasoactive sensory neuropeptides CGRP and substance P.

Neurokinin A (NKA) is a peptide neurotransmitter that participates in the regulation of breathing and the cardiovascular system. The purpose of the current study was to determine the cardiorespiratory pattern exerted by the systemic injection of NKA, to look at the contribution of neurokinin NK1 and NK2 receptors, and to establish the engagement of the vagal pathway in mediation of these responses. The effects of intravenous injections of NKA (50 μg/kg) were studied in anaesthetized, spontaneously breathing rats in the following experimental schemes: in neurally intact rats; and vagotomized at either midcervical or supranodosal level. Intravenous injections of NKA in the intact rats evoked sudden and short-lived increase in the respiratory rate concomitant with drop in tidal volume, followed by a prolonged depression, coupled with continuous augmentation of the tidal volume. Respiratory alterations were accompanied by transient tachycardia and prolonged hypotension. Midcervical vagotomy eliminated respiratory rate response and augmentation of tidal volume. Section of supranodosal vagi abrogated all respiratory reactions. NK2 receptor blockade abolished respiratory changes without affecting cardiovascular effects, whereas NK1 receptor blockade significantly reduced hypotension and increase in heart rate with no impact on the respiratory system. These results indicate that NKA induced changes in the breathing resulting from an excitation of the NK2 receptors on the vagal endings. A fall in blood pressure triggered by NKA occurs outside of the vagus nerve and is probably mediated via its direct action on vascular smooth muscles supplied with NK1 receptors.

The abnormal behaviour of NK1R-/- mice (locomotor hyperactivity, inattentiveness and impulsivity in the 5-Choice Serial Reaction-Time Test) is arguably analogous to that of patients with attention deficit hyperactivity disorder (ADHD). Evidence suggests that small body size and increased body weight are risk factors for ADHD. Here, we compared the body size, body mass and body composition of male and female NK1R-/- mice and their wildtypes that had been fed either standard laboratory chow or a high-fat (45%: 'Western') diet. Male NK1R-/- mice from both cohorts were approximately 7% shorter than wildtypes. A similar trend was evident in females. Male NK1R-/- mice fed the normal diet weighed less than wildtypes but the 'body mass index' ('mBMI': weight (mg)/length (cm)(2)) of female NK1R-/- mice was higher than wildtypes. When given the high-fat diet, the mBMI of both male and female NK1R-/- mice was higher than wildtypes. There were no consistent genotype or sex differences in protein, ash or water content of mice from the two cohorts. However, the fat content of male NK1R-/- mice on the Western diet was considerably (35%) higher than wildtypes and resembled that of females from both genotypes. We conclude that a lack of functional NK1R is associated with small body size but increases vulnerability to an increase in mBMI and fat content, especially in males. This phenotype could also be evident in ADHD patients with polymorphism(s) of the TACR1 gene (the human equivalent of Nk1r).

Antiemetic guideline recommendations are inconsistent as to whether a neurokinin-1 receptor antagonist (NK1 RA) should be administered with a 5-hydroxytryptamine-3 (5HT3) RA + dexamethasone (DEX) in patients receiving carboplatin. Patients receiving cisplatin routinely receive an NK1 RA-containing regimen with a resulting 14–22 % benefit in no emesis rates over a 5-HT3 RA/DEX control. Recent studies suggest a similar benefit in patients receiving carboplatin. NEPA is the first fixed antiemetic combination agent and comprises the highly selective NK1 RA, netupitant, and pharmacologically distinct 5-HT3 RA, palonosetron (PALO). This paper presents the efficacy of NEPA in the subset of patients receiving carboplatin in a phase 3 trial (NCT01376297), in the context of aprepitant (APR) data in the carboplatin setting. One hundred ninety-six patients (47 % of all study patients: n = 145 NEPA + DEX; n = 51 APR + PALO + DEX) received carboplatin in a multinational, double-blind, randomized phase 3 study. Complete response (CR: no emesis/rescue) and no significant nausea (NSN: score ≤25 on 100 mm visual analog scale) rates were calculated. Cycle 1–4 overall (0–120 h) CR rates were similar for NEPA (80, 91, 92, and 93 %) and APR (82, 88, 88, and 90 %). Overall NSN rates were also similar (NEPA 84–96 %; APR 82–90 %). Response rates for NEPA and APR regimens were similar and consistent with prior studies evaluating the contribution of adding NK1 RAs in patients receiving carboplatin. Considering such evidence, guideline groups/practitioners should consider giving a NK1 RA antiemetic triplet in patients receiving carboplatin.

Background Activation of capsaicin-sensitive sensory nerves and TRPV1 present on them can ameliorate the ischemia-reperfusion injury in vital organs by evoking the release of neuropeptides including calcitonin gene-related peptide (CGRP) and substance P. However, the underlying changes in TRPV1 and neuropeptide receptor expressions, including calcitonin receptor-like receptor (CRLR) and neurokinin 1 receptor (NK1R), after lung ischemia-reperfusion injury (LIRI) remain uncharacterized. Methods Thirty-two male Sprague–Dawley rats were randomly and equally divided into sham (sham thoracotomy) and ischemia-reperfusion (occlusion of the left pulmonary hilus for 1 h followed by reperfusion for 4 h) groups. Blood gas levels were measured and histopathologic examination was performed. Left lung lobes and brainstem tissue samples were harvested for use in quantitative real-time PCR, Western blot, and immunohistochemistry to measure TRPV1, CRLR, and NK1R transcript and protein levels. Additionally, CGRP and substance P protein levels were quantified in the lungs using enzyme-linked immunosorbent assay. Results LIRI exacerbated blood gas exchange and increased the pulmonary tissue injury score. Furthermore, LIRI increased CGRP levels in the lung, TRPV1–immunoreactivity (ir) in the bronchiolar epithelium and smooth muscle of the pulmonary artery, and the intensity of neuronal CRLR-ir and NK1R-ir in the commissural nucleus of the solitary tract. Similarly, LIRI significantly elevated both transcription and translation of TRPV1 in the lungs and CRLR and NK1R in the brainstem. Conclusions Both transcription and translation of TRPV1 in the lungs and CRLR and NK1R in the brainstem of rats can be upregulated by LIRI in a rapid manner (within 5 h).

Mast cell tryptase (MCT) is elevated in arthritic joints, but its direct effects are not known. Here, we investigated MCT-evoked acute inflammatory and nociceptive mechanisms with behavioural, in vivo imaging and immunological techniques. Neurogenic inflammation involving capsaicin-sensitive afferents, transient receptor potential vanilloid 1 receptor (TRPV1), substance P (SP), neurokinin A (NKA) and their NK1 tachykinin receptor were studied using gene-deleted mice compared to C57Bl/6 wildtypes (n = 5–8/group). MCT was administered intraarticularly or topically (20 μl, 12 μg/ml). Capsaicin-sensitive afferents were defunctionalized with the TRPV1 agonist resiniferatoxin (RTX; 30–70–100 μg/kg s.c. pretreatment). Knee diameter was measured with a caliper, synovial perfusion with laser Doppler imaging, mechanonociception with aesthesiometry and weight distribution with incapacitance tester over 6 h. Cytokines and neuropeptides were determined with immunoassays. MCT induced synovial vasodilatation, oedema, impaired weight distribution and mechanical hyperalgesia, but cytokine or neuropeptide levels were not altered at the 6-h timepoint. Hyperaemia was reduced in RTX-treated and TRPV1-deleted animals, and oedema was absent in NK1-deficient mice. Hyperalgesia was decreased in SP/NKA- and NK1-deficient mice, weight bearing impairment in RTX-pretreated, TRPV1- and NK1-deficient animals. MCT evokes synovial hyperaemia, oedema, hyperalgesia and spontaneous pain. Capsaicin-sensitive afferents and TRPV1 receptors are essential for vasodilatation, while tachykinins mediate oedema and pain.

Background Systemic administration of oxaliplatin has no effect on the tumors in the central nervous system (CNS) due to the limited concentration of oxaliplatin in the cerebrospinal fluid (CSF), while it was clinically reported that oxaliplatin can induce acute encephalopathy. Currently, the impairment of neuronal functions in the CNS after systemic administration of oxaliplatin remains uninvestigated. Methods The von Frey test and the plantar test were performed to evaluate neuropathic pain behavior after a single intraperitoneal administration of oxaliplatin (4 mg/kg) in rats. Inductively coupled plasma–mass spectrometry, electrophysiologic recording, real-time quantitative reverse transcription polymerase chain reaction, chromatin immunoprecipitation, Western blot, immunohistochemistry, and small interfering RNA were applied to understand the mechanisms. Results Concentration of oxaliplatin in CSF showed a time-dependent increase after a single administration of oxaliplatin. Spinal application of oxaliplatin at the detected concentration (6.6 nM) significantly increased the field potentials in the dorsal horn, induced acute mechanical allodynia (n = 12 each) and thermal hyperalgesia (n = 12 each), and enhanced the evoked excitatory postsynaptic currents and spontaneous excitatory postsynaptic currents in the projection neurokinin 1 receptor–expressing lamina I to II neurons. The authors further found that oxaliplatin significantly increased the nuclear factor-κB p65 binding and histone H4 acetylation in cx3cl1 promoter region. Thus, the upregulated spinal CX3CL1 markedly mediated the induction of central sensitization and acute pain behavior after oxaliplatin administration. Conclusions The findings of this study suggested that oxaliplatin in CSF may directly impair the normal function of central neurons and contribute to the rapid development of CNS-related side effects during chemotherapy. This provides novel targets to prevent oxaliplatin-induced acute painful neuropathy and encephalopathy.

The underlying cause(s) of abnormalities expressed by patients with attention deficit hyperactivity disorder (ADHD) have yet to be delineated. One factor that has been associated with increased vulnerability to ADHD is polymorphism(s) of TACR1, which is the human equivalent of the rodent NK1 (substance P-preferring) receptor gene (Nk1r). We have reported previously that genetically altered mice, lacking functional NK1R (NK1R-/-), express locomotor hyperactivity, which was blunted by the first-line treatment for ADHD, methylphenidate. Here, we compared the effects of this psychostimulant (3, 10 and 30 mg/kg, intraperitoneally) on the behaviour of NK1R-/- mice and their wild types in the 5-Choice Continuous Performance Test, which emulates procedures used to study attention and response control in ADHD patients. Methylphenidate increased total trials (a measure of 'productivity') completed by wild types, but not by NK1R-/- mice. Conversely, this drug reduced perseveration by NK1R-/- mice, but not by wild types. Other drug-induced changes in key behaviours were not genotype dependent, especially at the highest dose: for example, % omissions (an index of inattentiveness) was increased, whereas % false alarms and % premature responses (measures of impulsivity) declined in both genotypes, indicating reduced overall response. These findings are discussed in the context of the efficacy of methylphenidate in the treatment of ADHD. Moreover, they lead to several testable proposals. First, methylphenidate does not improve attention in a subgroup of ADHD patients with a functional deficit of TACR1. Second, these patients do not express excessive false alarms when compared with other groups of subjects, but they do express excessive perseveration, which would be ameliorated by methylphenidate.

Neuropilins are receptors for disparate ligands, including proangiogenic factors such as vascular endothelial growth factor and inhibitory class 3 semaphorin (SEMA3) family members. Differentiated cells in skin epithelium and cutaneous squamous cell carcinoma highly express the neuropilin-1 (NRP1) receptor. We examined the expression of NRP1 in human and mouse oral mucosa. NRP1 was significantly up-regulated in oral epithelial dysplasia and oral squamous cell carcinoma (OSCC). NRP1 receptor localized to the outer suprabasal epithelial layers in normal tongue, an expression pattern similar to the normal skin epidermis. However, dysplastic tongue epithelium and OSCC up-regulated NRP1 in basal and proliferating epithelial layers, a profile unseen in cutaneous squamous cell carcinoma. NRP1 up-regulation is observed in a mouse carcinogen-induced OSCC model and in human tongue OSCC biopsies. Human OSCC cell lines express NRP1 protein in vitro and in mouse tongue xenografts. Sites of capillary infiltration into orthotopic OSCC tumors correlate with high NRP1 expression. HSC3 xenografts, which express the highest NRP1 levels of the cell lines examined, showed massive intratumoral lymphangiogenesis. SEMA3A inhibited OSCC cell migration, suggesting that the NRP1 receptor was bioactive in OSCC. In conclusion, NRP1 is regulated in the oral epithelium and is selectively up-regulated during epithelial dysplasia. NRP1 may function as a reservoir to sequester proangiogenic ligands within the neoplastic compartment, thereby recruiting neovessels toward tumor cells.

Cytoplasmic calcium (Ca(2+)) mobilization has been proposed to be an important factor in the induction of emesis. The selective sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor thapsigargin, is known to deplete intracellular Ca(2+) stores, which consequently evokes extracellular Ca(2+) entry through cell membrane-associated channels, accompanied by a prominent rise in cytosolic Ca(2+). A pro-drug form of thapsigargin is currently under clinical trial as a targeted cancer chemotherapeutic. We envisioned that the intracellular effects of thapsigargin could cause emesis and planned to investigate its mechanisms of emetic action. Indeed, thapsigargin did induce vomiting in the least shrew in a dose-dependent and bell-shaped manner, with maximal efficacy (100%) at 0.5 mg/kg (i.p.). Thapsigargin (0.5 mg/kg) also caused increases in c-Fos immunoreactivity in the brainstem emetic nuclei including the area postrema (AP), nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus (DMNX), as well as enhancement of substance P (SP) immunoreactivity in DMNX. In addition, thapsigargin (0.5 mg/kg, i.p.) led to vomit-associated and time-dependent increases in phosphorylation of Ca(2+)/calmodulin kinase IIα (CaMKIIα) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) in the brainstem. We then explored the suppressive potential of diverse chemicals against thapsigargin-evoked emesis including antagonists of: i) neurokinin-1 receptors (netupitant), ii) the type 3 serotonin receptors (palonosetron), iii) store-operated Ca(2+) entry (YM-58483), iv) L-type Ca(2+) channels (nifedipine), and v) SER Ca(2+)-release channels inositol trisphosphate (IP3Rs) (2-APB)-, and ryanodine (RyRs) (dantrolene)-receptors. In addition, the antiemetic potential of inhibitors of CaMKII (KN93) and ERK1/2 (PD98059) were investigated. All tested antagonists/blockers attenuated emetic parameters to varying degrees except palonosetron, however a combination of non-effective doses of netupitant and palonosetron exhibited additive antiemetic efficacy. A low-dose combination of nifedipine and 2-APB plus dantrolene mixture completely abolished thapsigargin-evoked vomiting, CaMKII-ERK1/2 activation and SP elevation. In addition, pretreatment with KN93 or PD98059 suppressed thapsigargin-induced increases in SP and ERK1/2 activation. Intracerebroventricular injection of netupitant suppressed vomiting caused by thapsigargin which suggests that the principal site of evoked emesis is the brainstem. In sum, this is the first study to demonstrate that thapsigargin causes vomiting via the activation of the Ca(2+)-CaMKII-ERK1/2 cascade, which is associated with an increase in the brainstem tissue content of SP, and the evoked emesis occurs through SP-induced activation of neurokinin-1 receptors.

Chemotherapy-induced nausea and vomiting (CINV), both acute and delayed, has a dramatic effect on the well-being and quality of life of patients with cancer. Improved understanding of the mechanisms involved in CINV has led to the development of agents targeting the 5-HT3 receptor as well as the NK-1 receptor. Antiemetic prophylaxis given to patients receiving highly emetogenic chemotherapy combines agents blocking the 5-HT3 and NK-1 receptors along with corticosteroids given regularly and repeatedly. Rolapitant is a long-acting NK-1 receptor antagonist with proven efficacy in controlling CINV as part of the prophylaxis regimen. This review will detail the clinical efficacy and safety of rolapitant in the treatment of patients with cancer receiving highly or moderately emetogenic chemotherapy.

The mechanisms by which G proteins modulate voltage-gated Ca2+ channel currents (CaV), particularly CaV2.2 and CaV2.3, are voltage dependent (VD) or voltage independent (VI). VD pathways are typically mediated by Gαi/o and GαS subfamilies. On the other hand, VI inhibition modulation is coupled to the Gαq subfamily and signaling pathways downstream of phospholipase C stimulation. In most studies, this latter pathway has been shown to be linked to Gαq and/or Gα11 protein subunits. However, there are no studies that have examined whether natively expressed Gα14 subunits (Gαq subfamily member) couple G protein-coupled receptors (GPCR) with CaV2.2 channels. We report that Gα14 subunits functionally couple the substance P (SP)/neurokinin-1 (NK-1) receptor pathway to CaV2.2 channels in acutely dissociated rat celiac-superior mesenteric ganglion (CSMG) neurons. Exposure of CSMG neurons to SP blocked the CaV2.2 currents in a predominantly VD manner that was pertussis toxin and cholera toxin resistant, as well as Gαq/11 independent. However, silencing Gα14 subunits significantly attenuated the SP-mediated Ca2+ current block. In another set of experiments, exposure of CSMG neurons to SP led to the inhibition of KCNQ K+ M-currents. The SP-mediated M-current block was significantly reduced in neurons transfected with Gα14 small-interference RNA. Finally, overexpression of the GTP-bound Gαq/11 binding protein RGS2 did not alter the block of M-currents by SP but significantly abolished the oxotremorine methiodide-mediated M-current inhibition. Taken together, these results provide evidence of a new Gα14-coupled signaling pathway that modulates CaV2.2 and M-currents via SP-stimulated NK-1 receptors in CSMG neurons.

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 nAChRs 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.

Noxious stimuli activate small to medium-sized dorsal root ganglion (DRG) neurons. Intense noxious stimuli result in the release of substance P (SP) from the central terminals of these neurons. It binds to the neurokinin type 1 receptor (NK1r) and sensitises the dorsal horn neurons. SP is also released from the peripheral terminals leading to neurogenic inflammation. However, their individual contribution at spinal and peripheral levels to postincisional nociception has not been delineated as yet.Sprague-Dawley rats were administered different doses (3-100 μg) of an NK1r antagonist (L760735) by intrathecal (i.t.) route before hind paw incision. On the basis of its antinociceptive effect on guarding behaviour, the 30 μg dose was selected for further study. In different sets of animals, this was administered i.t. (postemptive) and intrawound (i.w.). Finally, in another group, drug (30 μg) was administered through both i.t and i.w. routes. The antinociceptive effect was assessed and compared. Expression of SP was examined in the spinal cord. Intrawound concentration of SP and inflammatory mediators was also evaluated.Postemptive i.t. administration significantly attenuated guarding and allodynia. Guarding was alone decreased after i.w. drug treatment. Combined drug administration further attenuated all nociceptive parameters, more so after postemptive treatment. Expression of SP in the spinal cord decreased post incision but increased in the paw tissue. Inflammatory mediators like the nerve growth factor also increased after incision.In conclusion, SP acting through the NK1r appears to be an important mediator of nociception, more so at the spinal level. These findings could have clinical relevance.

We have previously shown that cough potentiation induced by intravenous administration of the AT 1 receptor antagonist losartan is lower than that induced by the ACE inhibitor lisinopril in anesthetized and awake rabbits. Since losartan and lisinopril cross the blood–brain barrier, their central action on the cough reflex can be hypothesized. Mechanical stimulation of the tracheobronchial tree and citric acid inhalation were used to induce cough reflex responses in pentobarbital sodium-anesthetized, spontaneously breathing rabbits. Bilateral microinjections (30–50 nl) of losartan (5 mM), lisinopril (1 mM), bradykinin (0.05 mM), HOE-140 (0.2 mM, a bradykinin B 2 receptor antagonist) and CP-99,994 (1 mM, an NK 1 receptor antagonist) were performed into the caudal nucleus tractus solitarii, the predominant site of termination of cough-related afferents. Lisinopril, but not losartan increased the cough number. This effect was reverted by HOE-140 or CP-99,994. Cough potentiation was also induced by bradykinin. The results support for the first time a central protussive action of lisinopril mediated by an accumulation of bradykinin and substance P.

The substance P (SP)/NK-1 receptor (NK1R) complex represents an intriguing anticancer target for a variety of tumors, including hepatoblastoma (HB). Therefore, NK1R antagonists, such as the clinical drug aprepitant, recently have been proposed as potent anticancer agents. However, very little is known regarding the molecular basis of NK1R inhibition in cancer. Using reverse phase protein array, Western blot, Super TOP/FOP, confocal microscopy, and sphere formation ability (SFA) assays, we identified the AKT and Wnt signaling pathways as the key targets of aprepitant in three human HB cell lines (HepT1, HepG2, and HuH6). Following NK1R blockage, we observed decreased phosphorylation of p70S6K and 4E-BP1/2 and inhibition of the canonical Wnt pathway with subsequent decrease of HB cell growth. This effect was dependent of high baseline Wnt activity either by mutational status of β-catenin or extrinsic Wnt activation. Wnt inhibition seemed to be strengthened by disruption of the FOXM1–β-catenin complex. Furthermore, treatment of HB cells with aprepitant led to reduced expression of (liver) stemness markers (AFP, CD13, SOX2, NANOG, and OCT4) and SFA when grown under cancer stem cell conditions. Taken together, we show for the first time that targeting the SP/NK1R signaling cascade inhibits canonical Wnt signaling in HB cells. These findings reveal important insight into the molecular mechanisms of the SP/NK1R complex as a critical component in a model of pediatric liver cancer and may support the development of novel therapeutic interventions for HB and other Wnt-activated cancers. Mol Cancer Ther; 14(12); 2712–21. ©2015 AACR.

The brain serotonergic system is colocalized and interacts with the neuropeptidergic substance P/neurokinin-1 (SP/NK1) system. Both these neurochemical systems have independently been implicated in stress and anxiety, but interactions between them might be crucial for human anxiety conditions. Here, we examined the serotonin and substance P/neurokinin-1 (SP/NK1) systems individually as well as their overlapping expression in 16 patients with posttraumatic stress disorder (PTSD) and 16 healthy controls. Participants were imaged with the highly selective radiotracers [(11)C]-3-amino-4-(2-dimethylaminomethylphenylsulfanyl)-benzonitrile (DASB) and [(11)C]GR205171 assessing serotonin transporter (SERT) and NK1 receptor availability, respectively. Voxel-wise analyses in the amygdala, our a priori-defined region of interest, revealed increased number of NK1 receptors, but not SERT in the PTSD group. Symptom severity, as indexed by the Clinician-administered PTSD Scale, was negatively related to SERT availability in the amygdala, and NK1 receptor levels moderated this relationship. Exploratory, voxel-wise whole-brain analyses revealed increased SERT availability in the precentral gyrus and posterior cingulate cortex of PTSD patients. Patients, relative to controls, displayed lower degree of overlapping expression between SERT and NK1 receptors in the putamen, thalamus, insula and lateral orbitofrontal gyrus, lower overlap being associated with higher PTSD symptom severity. Expression overlap also explained more of the symptomatology than did either system individually, underscoring the importance of taking interactions between the neurochemical systems into account. Thus, our results suggest that aberrant serotonergic-SP/NK1 couplings contribute to the pathophysiology of PTSD and, consequently, that normalization of these couplings may be therapeutically important.

Background: Neurotransmitters, including substance P (SP) and neurokinin A (NKA), are widely distributed in both the central and peripheral nervous system and their receptors, neurokinin-1 receptor (NK1R) and neurokinin-2 receptor (NK2R), are expressed on immune cells. However, the role of the NKA-NK2R axis in immune responses relative to the SP-NK1R signaling cascade has not been elucidated. Objective: We sought to examine the effect of neuropeptide signaling through NK1Rand NK2R on antigen presentation by dendritic cells (DCs) and the subsequent activation of effector Th cells. Methods: Expression levels of NK1R, NK2R, HLA-class II and costimulatory molecules of human MoDCs and cytokine production by birch pollen antigen-specific CD4+ T cells cocultured with MoDCs in the presence of NK1R and NK2R antagonists were evaluated by quantitative RT-PCR, flow cytometry or ELISA. NK1R and NK2R expression in the lung of patients with asthma and hypersensitivity pneumonitis was evaluated by immunohistochemistry. Results: Human MoDCs significantly upregulated NK2R and NK1R expression in response to poly I:C stimulation in a STAT1-dependent manner. Both NK2R and NK1R were expressed on alveolar macrophages and lung DCs from patients with asthma and pneumonitis hypersensitivity. Surface expression levels of HLA-class II and costimulatory molecules on DCs were modulated by NK1R or NK2R antagonists. Activation of birch pollen-derived antigen-specific CD4+ T cells and their production of cytokines including IL-4 and IFN-γ as well as IL-12 production by MoDCs, were suppressed by blocking NK1R or NK2R after in vitro antigen stimulation. Conclusions: NK1R- and NK2R-mediated neuropeptide signaling promotes both innate and acquired immune responses through activation of human DCs.

Latent sensitization is a model of chronic pain in which a persistent state of pain hypersensitivity is suppressed by opioid receptors, as evidenced by the ability of opioid antagonists to induce a period of mechanical allodynia. Our objective was to determine if substance P and its neurokinin 1 receptor (NK1R) mediate the maintenance of latent sensitization. Latent sensitization was induced by injecting rats in the hindpaw with complete Freund's adjuvant (CFA), or by tibial spared nerve injury (SNI). When responses to von Frey filaments returned to baseline (day 28), the rats were injected intrathecally with saline or the NK1R antagonist RP67580, followed 15 min later by intrathecal naltrexone. In both pain models, the saline-injected rats developed allodynia for 2 h after naltrexone, but not the RP67580-injected rats. Saline or RP67580 were injected daily for two more days. Five days later (day 35), naltrexone was injected intrathecally. Again, the saline-injected rats, but not the RP67580-injected rats, developed allodynia in response to naltrexone. To determine if there is sustained activation of NK1Rs during latent sensitization, NK1R internalization was measured in lamina I neurons in rats injected in the paw with saline or CFA, and then injected intrathecally with saline or naltrexone on day 28. The rats injected with CFA had a small amount of NK1R internalization that was significantly higher than in the saline-injected rats. Naltrexone increased NK1R internalization in the CFA-injected rats but nor in the saline-injected rats. Therefore, sustained activation of NK1Rs maintains pain hypersensitivity during latent sensitization.

Objectives TGFβ promotes cancer aggressiveness in advanced stages. NK1R-Tr expression in advanced breast cancer has a pro-carcinogenic effect. In this study, we aimed to investigate the effect of the association of TGFβ with NK1R-Tr expression on the proliferation and apoptosis of breast cancer cells. Methods Immunohistochemical staining and Western blot analysis were used to detect TGFβ and NK1R-Tr in breast cancer and paracancerous tissue samples. MDA-MB-231 and BT549 cells were stimulated with TGFβ after NK1R knockdown or treated with the NK1R antagonist aprepitant, and the effects of TGFβ and NK1R-Tr on proliferation and apoptosis were detected by CCK-8, colony formation and flow cytometry assays. In vivo xenograft models were used to further verify the effects of NK1R-Tr and TGFβ. The regulatory effects of Smad4 on NK1R promoter activity were confirmed by ChIP and dual-luciferase reporter assays. Results The expression levels of TGFβ and NK1R-Tr were higher in breast cancer tissues than in adjacent tissues and were positively correlated in human breast cancer tissues. NK1R knockdown or aprepitant treatment in MDA-MB-231 and BT549 cells attenuated the effects of TGFβ on cell proliferation. The proportion of cells in G2/M phase significantly increased, the expression of cyclin B1 decreased, and the expression of P21 increased; these effects were weakened by TGFβ treatment. Apoptosis in breast cancer cells was significantly increased. In vivo xenograft models were used to further verify that NK1R-Tr and TGFβ promoted tumour growth. After TGFβ treatment, the binding capacity of Smad4 to the NK1R promoter, as well as luciferase activity, was enhanced. Conclusions The expression levels of TGFβ and NK1R-Tr were higher in breast cancer tissues than in normal tissues, and both were correlated with a poor patient prognosis. TGFβ and NK1R-Tr promoted cell proliferation and inhibited apoptosis, and TGFβ regulated the expression of NK1R-Tr via Smad4.

Previous studies have demonstrated that continuous substance P (SP) infusion into the rat striatum attenuated hind paw formalin-induced nociceptive behaviors and mechanical hypersensitivity via a neurokinin-1 (NK1) receptor dependent mechanism. However, whether there is a role of striatal infusion of SP on chronic, neuropathic pain has yet to be demonstrated. The present study investigated the effect of continuous SP infusion into the rat striatum using a reverse microdialysis method is antinociceptive in a rat model of chronic, mononeuropathic pain. Two weeks after partial sciatic nerve injury, the ipsilateral hind paw demonstrated mechanical hypersensitivity. Infusion of SP (0.2, 0.4, or 0.8 μg/mL, 1 μL/min) for 120 min into the contralateral striatum dose-dependently relieved mechanical hypersensitivity. The antinociceptive effect of SP infusion was inhibited by co-infusion with the NK1 receptor antagonist CP96345 (10 μM). Neither ipsilateral continuous infusion nor acute microinjection of SP (10 ng) into the contralateral striatum was antinociceptive. A role of striatal muscarinic cholinergic neurons is suggested since co-infusion of SP with atropine (10 μM), but not the nicotinic receptor mecamylamine (10 μM), blocked antinociception. The current study suggests that activation of striatal muscarinic receptors through NK1 receptors could be a novel approach to managing chronic pain.

Purpose Neurotrophic keratopathy is a corneal epitheliopathy induced by trigeminal denervation that can be treated with eyedrops containing the neuropeptide substance P (or the peptide FGLM-NH2 derived therefrom) and insulin-like growth factor 1 (or the peptide SSSR derived therefrom). Here, we examine the mechanism by which substance P (or FGLM-NH2) promotes corneal epithelial wound healing in a mouse model of neurotrophic keratopathy. Methods The left eye of mice subjected to trigeminal nerve axotomy in the right eye served as a model of neurotrophic keratopathy. Corneal epithelial wound healing was monitored by fluorescein staining and slit-lamp examination. The distribution of substance P, neurokinin-1 receptor (NK-1R), and phosphorylated Akt was examined by immunohistofluorescence analysis. Cytokine and chemokine concentrations in intraocular fluid were measured with a multiplex assay. Results Topical administration of FGLM-NH2 and SSSR promoted corneal epithelial wound healing in the neurotrophic keratopathy model in a manner sensitive to the NK-1R antagonist L-733,060. Expression of substance P and NK-1R in the superficial layer of the corneal epithelium decreased and increased, respectively, in model mice compared with healthy mice. FGLM-NH2 and SSSR treatment suppressed the production of interleukin-1α, macrophage inflammatory protein 1α (MIP-1α) and MIP-1β induced by corneal epithelial injury in the model mice. It also increased the amount of phosphorylated Akt in the corneal epithelium during wound healing in a manner sensitive to prior L-733,060 administration. Conclusions The substance P-NK-1R axis promotes corneal epithelial wound healing in a neurotrophic keratopathy model in association with upregulation of Akt signaling and attenuation of changes in the cytokine-chemokine network.

Anaphylaxis during general anaesthesia is a significant clinical challenge for anaesthesiologists. Approximately 50% of perioperative anaphylaxis cases lack the presence of specific IgE antibodies. Mas-related G-protein coupled receptor X2 (MRGPRX2) in humans and its mouse orthologue Mas-related G-protein coupled receptor B2 (Mrgprb2) are crucial receptors in non-IgE-dependent histamine release. Anaesthetics such as rocuronium and atracurium cause perioperative anaphylaxis by activating histamine release via the Mrgprb2 pathway. We hypothesized that antagonistic DNA aptamers that target MRGPRX2 can prevent perioperative anaphylaxis. Selection of a DNA aptamer that specifically binds MRGPRX2 was achieved by using our modified Systematic Evolution of Ligands by Exponential enrichment (SELEX) approach. Our SELEX process used MRGPRX2-proteoliposomes synthesised by a wheat germ cell-free system as templates. The activity of the selected aptamer to inhibit histamine release from MRGPRX2-activated mast cells and in an anaphylaxis rat model transplanted with this cell line was examined. Our selection process identified aptamer-X35 with the sequence 5′-ATGACCATGACCCTCCACACTGTAGGCACCACGGGTCCCTGGCAGTTAAAAGTACGTTTGTCAGACTGTGGCAGGGAAACA-3'. In silico 2D modelling of aptamer-X35 revealed a structure with a small loop and a long stem. Aptamer-X35 inhibited histamine release from mast cells by 70%. Subcutaneous injection of 30 nmol of aptamer-X35 inhibited the anaphylactic reaction in the rat anaphylaxis model. This study demonstrated that aptamer-X35 selected by the modified SELEX approach reduced histamine release by inhibiting the MRGPRX2 pathway. Overall, our findings establish aptamer-X35 as a potential therapeutic candidate against perioperative anaphylaxis.

SUMMARY Efficient Ca2+ flux induced during cognate T cell activation requires signaling the T cell receptor (TCR) and unidentified G-protein-coupled receptors (GPCRs). T cells express the neurokinin-1 receptor (NK1R), a GPCR that mediates Ca2+ flux in excitable and non-excitable cells. However, the role of the NK1R in TCR signaling remains unknown. We show that the NK1R and its agonists, the neuropeptides substance P and hemokinin-1, co-localize within the immune synapse during cognate activation of T cells. Simultaneous TCR and NK1R stimulation is necessary for efficient Ca2+ flux and Ca2+-dependent signaling that sustains the survival of activated T cells and helper 1 (Th1) and Th17 bias. In a model of contact dermatitis, mice with T cells deficient in NK1R or its agonists exhibit impaired cellular immunity, due to high mortality of activated T cells. We demonstrate an effect of the NK1R in T cells that is relevant for immunotherapies based on pro-inflammatory neuropeptides and its receptors., Graphical Abstract, In Brief The neurokinin 1 receptor (NK1R) induces Ca2+ flux in excitable cells. Here, Morelli et al. show that NK1R signaling in T cells promotes optimal Ca2+ flux triggered by TCR stimulation, which is necessary to sustain T cell survival and the efficient Th1- and Th17-based immunity that is relevant for immunotherapies based on pro-inflammatory neuropeptides.