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The neurokinin-1 receptor (NK1R; encoded by Tacr1 ) is expressed in spinal dorsal horn neurons and has been suggested to mediate itch in rodents. However, previous studies relied heavily on neurotoxic ablation of NK1R spinal neurons, which limited further dissection of their function in spinal itch circuitry. To address this limitation, we leveraged a newly developed Tacr1 CreER mouse line to characterize the role of NK1R spinal neurons in itch. We show that pharmacological activation of spinal NK1R and chemogenetic activation of Tacr1 CreER spinal neurons increases itch behavior in male and female mice, whereas pharmacological inhibition of spinal NK1R suppresses itch behavior. We use fluorescence in situ hybridization (FISH) to characterize the endogenous expression of Tacr1 throughout the superficial and deeper dorsal horn (DDH), as well as the lateral spinal nucleus (LSN), of mouse and human spinal cord. Retrograde labeling studies in mice from the parabrachial nucleus (PBN) show that less than 20% of superficial Tacr1 CreER dorsal horn neurons are spinal projection neurons, and thus the majority of Tacr1 CreER are local interneurons. We then use a combination of in situ hybridization and ex vivo two-photon Ca 2+ imaging of the mouse spinal cord to establish that NK1R and the gastrin-releasing peptide receptor (GRPR) are coexpressed within a subpopulation of excitatory superficial dorsal horn (SDH) neurons. These findings are the first to suggest a role for NK1R interneurons in itch and extend our understanding of the complexities of spinal itch circuitry. SIGNIFICANCE STATEMENT The spinal cord is a critical hub for processing somatosensory input, yet which spinal neurons process itch input and how itch signals are encoded within the spinal cord is not fully understood. We demonstrate neurokinin-1 receptor (NK1R) spinal neurons mediate itch behavior in mice and that the majority of NK1R spinal neurons are local interneurons. These NK1R neurons comprise a subset of gastrin-releasing peptide receptor (GRPR) interneurons and are thus positioned at the center of spinal itch transmission. We show NK1R mRNA expression in human spinal cord, underscoring the translational relevance of our findings in mice. This work is the first to suggest a role for NK1R interneurons in itch and extends our understanding of the complexities of spinal itch circuitry., (Copyright © 2020 the authors.)

Mitochondria, as primary energy generators and Ca 2+ 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-Bötzinger complex (pre-BötC). The metabolic marker of neuronal activity, cytochrome c oxidase (CO), and dendritic mitochondria were examined in pre-BötC 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-BötC 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-BötC 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-BötC responded dynamically to respiratory plasticity. Hence, plastic neuronal changes are closely coupled to active mitochondrial bioenergetics, leading to enhanced energy production and Ca 2+ buffering that may drive the LTF expression., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Uncontrollable itch-scratching cycles lead to serious skin damage in patients with chronic itch. However, the neural mechanism promoting the itch-scratching cycle remains elusive. Here, we report that tachykinin 1 (Tac1)-expressing glutamatergic neurons in the lateral and ventrolateral periaqueductal gray (l/vlPAG) facilitate the itch-scratching cycle. We found that l/vlPAG neurons exhibited scratching-behavior-related neural activity and that itch-evoked scratching behavior was impaired after suppressing the activity of l/vlPAG neurons. Furthermore, we showed that the activity of Tac1-expressing glutamatergic neurons in the l/vlPAG was elevated during itch-induced scratching behavior and that ablating or suppressing the activity of these neurons decreased itch-induced scratching behavior. Importantly, activation of Tac1-expressing neurons induced robust spontaneous scratching and grooming behaviors. The scratching behavior evoked by Tac1-expressing neuron activation was suppressed by ablation of spinal neurons expressing gastrin-releasing peptide receptor (GRPR), the key relay neurons for itch. These results suggest that Tac1-expressing neurons in the l/vlPAG promote itch-scratching cycles., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objectives: MiR-34 is a tumour suppressor in breast cancer. Neurokinin-1 receptor (NK1R), which is the predicted target of the miR-34 family, is overexpressed in many cancers. This study investigated the correlation and clinical significance of miR-34 and NK1R in breast cancer., Materials and Methods: Western blotting, quantitative reverse transcription-PCR (qRT-PCR) and luciferase assays were conducted to analyse the regulation of NK1R by miR-34 in MDA-MB-231, MCF-7, T47D, SK-BR-3 and HEK-293 T cells. MiR-34b/c-5p, full-length NK1R (NK1R-FL) and truncated NK1R (NK1R-Tr) expression in fifty patients were quantified by qRT-PCR and correlated with their clinicopathological parameters. CCK-8 assays, colony formation assays and flow cytometry were used to measure cell proliferation and apoptosis in MDA-MB-231 and MCF-7 cells transfected with miR-34b/c-5p or NK1R-siRNA and before treatment with or without Substance P (SP), an endogenous peptide agonists of NK1R. The effect of NK1R antagonist aprepitant was also investigated. In vivo xenograft models were used to further verify the regulation of NK1R by miR-34b/c-5p., Results: Expression levels of miR-34b/c-5p and NK1R-Tr, but not NK1R-FL, were associated with enhanced malignant potential, such as tumour stage and Ki67 expression. The overexpression of miR-34b/c-5p or NK1R silencing potently suppressed cell proliferation and induced G2/M phase arrest and the apoptosis of MDA-MB-231 and MCF-7 cells. The NK1R antagonist aprepitant had similar effects. In vivo studies confirmed that miR-34b/c-5p overexpression or NK1R silencing reduced the tumorigenicity of breast cancer. In addition, SP rescued the effects of miR-34b/c-5p overexpression or NK1R silencing on cell proliferation and apoptosis in vitro and in vivo assays., Conclusions: MiR-34b/c-5p and NK1R contribute to breast cancer cell proliferation and apoptosis and are potential targets for breast cancer therapeutics., (© 2018 The Authors Cell Proliferation Published by John Wiley & Sons Ltd.)

Rationale: Although alcohol use disorder and anxiety disorders are highly comorbid in humans, controversy remains regarding whether anxiety predisposes individuals to alcohol reward, and the relationship with neurokinin-1 receptor (NK1R) is unclear., Objectives: The objectives of the study are to investigate the association between anxiety-like behavior and alcohol-induced conditioned place preference (CPP) and to examine the effect of NK1R antagonist L-703,606 on this preference and levels of NK1R protein in different brain regions in adolescent mice., Methods: The anxiety-like behavior of adolescent male C57BL/6 mice was assessed using the elevated plus maze (EPM) test, and the animals were then allocated into high-anxiety mouse (HAM) and low-anxiety mouse (LAM) groups based on the percent of open arm time (OT%). After the reinforcement of ethanol was established by alcohol-induced CPP (2 g/kg), NK1R expression was quantified in the hippocampus, prefrontal cortex, and amygdala. Finally, the effect of L-703,606 (10 mg/kg) on the alcohol-induced CPP was examined., Results: LAM showed a greater ethanol preference (P = 0.004) and a higher level of NK1R protein in the hippocampus (P = 0.026) than HAM group. Interestingly, the CPP score positively correlated with OT% (r = 0.520, P = 0.016) and the level of NK1R protein (r = 0.476, P = 0.029) in the hippocampus. Moreover, L-703,606 attenuated alcohol-induced CPP (P < 0.001) in both groups., Conclusions: The present results highlight the negative correlation between anxiety-like behavior and the propensity for alcohol and the critical role for NK1R in alcohol reward in adolescent mice. Importantly, the NK1R antagonist L-703,606 might be a promising therapeutic target for alcohol use disorder.

Neuropathic pain in a chronic post-ischaemic pain (CPIP) model mimics the symptoms of complex regional pain syndrome type I (CRPS I). The administration of bee venom (BV) has been utilized in Eastern medicine to treat chronic inflammatory diseases accompanying pain. However, the analgesic effect of BV in a CPIP model remains unknown. The application of a tight-fitting O-ring around the left ankle for a period of 3 h generated CPIP in C57/Bl6 male adult mice. BV (1 mg/kg ; 1, 2, and 3 times) was administered into the SC layer of the hind paw, and the antiallodynic effects were investigated using the von Frey test and by measuring the expression of neurokinin type 1 (NK-1) receptors in dorsal root ganglia (DRG). The administration of BV dose-dependently reduced the pain withdrawal threshold to mechanical stimuli compared with the pre-administration value and with that of the control group. After the development of the CPIP model, the expression of NK-1 receptors in DRG increased and then decreased following the administration of BV. SC administration of BV results in the attenuation of allodynia in a mouse model of CPIP. The antiallodynic effect was objectively proven through a reduction in the increased expression of NK-1 receptors in DRG.

Substance P (SP) was reported to be associated with eczema and acts as a potent skin mast cell secretagogue. However, little is known of its expression in inflammatory cells in eczema and its ability in induction of mast cell accumulation. In the present study, we investigated expression of SP and neurokinin-1 receptor (NK1R) on peripheral blood leukocytes and mast cells from patients with eczema and influence of SP on mast cell accumulation by using flow cytometry analysis, trans-epithelial cell migration assay and mouse peritoneal model. The results showed that plasma SP and IL-17A levels in eczema patients were higher than that in healthy control subject. The percentages of SP+ and NK1R+ expression populations of monocytes, helper T cells, natural killer T cells and basophils in peripheral blood of eczema patients were markedly elevated. It was observed that not only absolute number of mast cells but also SP+ and NK1R+ mast cells are enhanced in the lesion skin of eczema. SP showed a potent chemoattractant action on mast cells as assessed by a mouse peritoneal model and a trans-endothelium cell migration assay. SP-induced mast cell accumulation appears a CD18/CD11a complex, L-selectin and ICAM-1-dependent event which can be blocked by a NK-1R antagonist RP67580. In conclusion, elevated expression of SP in patients with eczema and the ability of SP in induction of mast cell accumulation indicate strongly that SP is a potent proinflammatory mediator, which contributes to the pathogenesis of eczema. Inhibitors of SP and blockers of NK1R are likely useful agents for treatment of eczema.

Purpose: Transforming growth factor beta 1 (TGF-β1) is a cytokine involved in a variety of processes, such as differentiation of fibroblasts into myofibroblasts. TGF-β1 has also been shown to delay the internalization of the neurokinin-1 receptor (NK-1 R) after its activation by its ligand, the neuropeptide substance P (SP). NK-1 R comprises two naturally occurring variants, a full-length and a truncated form, triggering different cellular responses. SP has been shown to affect important events in the cornea - such as stimulating epithelial cell proliferation - processes that are involved in corneal wound healing and thus in maintaining the transparency of the corneal stroma. An impaired signaling through NK-1 R could thus impact the visual quality. We hypothesize that TGF-β1 modulates the expression pattern of NK-1 R in human corneal stroma cells, keratocytes. The purpose of this study was to test that hypothesis., Methods: Cultures of primary keratocytes were set up with cells derived from healthy human corneas, obtained from donated transplantation graft leftovers, and characterized by immunocytochemistry and Western blot. Immunocytochemistry for TGF-β receptors and NK-1 R was performed. Gene expression was assessed with real-time polymerase chain reaction (qPCR)., Results: Expression of TGF-β receptors was confirmed in keratocytes in vitro. Treating the cells with TGF-β1 significantly reduced the gene expression of NK-1 R. Furthermore, immunocytochemistry for NK-1 R demonstrated that it is specifically the expression of the full-length isotype of the receptor that is reduced after treatment with TGF-β1, which was also confirmed with qPCR using a specific probe for the full-length receptor., Conclusions: TGF-β1 down-regulates the gene expression of the full-length variant of NK-1 R in human keratocytes, which might impact its signaling pathway and thus explain the known delay in internalization after activation by SP seen with TGF-β1 treatment.

Objectives: It has been demonstrated that substance P (SP) promotes while neurokinin-1 receptor (NK-1R) antagonist inhibits the proliferation of several human cancer cells. Currently, it is still unknown whether such actions exist in human endometrial carcinoma. This study aimed to explore the role of SP/NK-1R signaling in the progression of endometrial adenocarcinoma., Materials and Methods: The expression levels of SP and NK-1R in endometrial adenocarcinoma tissues and Ishikawa cell line were detected by real-time quantitative PCR and Western blot analysis. The effects of SP on Ishikawa cells proliferation and invasion were analyzed using MTT assay and transwell matrigel invasion assay, respectively. The expression levels of matrix metalloproteinase 9 (MMP-9) and vascular endothelial growth factor C (VEGF-C) in Ishikawa cells after administration of SP were detected by real-time quantitative RCR and Western blot analysis., Results: The expression levels of SP and NK-1R were significantly higher in endometrial adenocarcinoma tissues and Ishikawa cells than in normal endometrium. Substance P significantly enhanced the proliferation and invasion of Ishikawa cells. In addition, SP induced the expression of MMP-9 and VEGF-C in Ishikawa cells, whereas NK-1R antagonist inhibited these effects., Conclusions: Substance P plays an important role in the development of endometrial carcinoma by inducing the expression of MMP-9 and VEGF-C and promoting cancer cell proliferation and metastasis, which can be blocked by NK-1R antagonist.

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., (©2015 American Association for Cancer Research.)

The peptides of the tachykinin family participate in the regulation of reproductive function acting at both central and peripheral levels. Our previous data showed that treatment of rats with a tachykinin NK3R antagonist caused a reduction of litter size. In the present study, we analyzed the expression of tachykinins and tachykinin receptors in the rat uterus during early pregnancy. Uterine samples were obtained from early pregnant rats (Days 1-9 of pregnancy) and from nonpregnant rats during the proestrus stage of the ovarian cycle, and real-time quantitative RT-PCR, immunohistochemistry, and Western blot studies were used to investigate the pattern of expression of tachykinins and tachykinin receptors. We found that all tachykinins and tachykinin receptors were locally synthesized in the uterus of early pregnant rats. The expression of substance P, neurokinin B, and the tachykinin receptors NK1R and NK3R mRNAs and proteins underwent major changes during the days around implantation and they were widely distributed in implantation sites, being particularly abundant in decidual cells. These findings support the involvement of the tachykinin system in the series of uterine events that occur around embryo implantation in the rat., (© 2015 by the Society for the Study of Reproduction, Inc.)

We investigated roles for spinal neurons expressing the neurokinin-1 receptor (NK1R) and/or gastrin-releasing peptide receptor (GRPR) in a mouse model of ovalbumin (OVA)-induced chronic atopic dermatitis. Mice receiving repeated topical application of OVA exhibited atopic-like skin lesions and behavioral signs of chronic itch including spontaneous scratching, touch-evoked scratching (alloknesis), and enhancement of chloroquine-evoked scratching (hyperknesis). Substance P-saporin (SP-SAP) and bombesin-saporin (BB-SAP) were intrathecally injected into OVA-sensitized mice to neurotoxically ablate NK1R- or GRPR-expressing spinal neurons, respectively. SP-SAP diminished the expression of NK1R in the superficial spinal dorsal horn and significantly attenuated all behavioral signs of chronic itch. BB-SAP reduced the spinal dorsal horn expression of GRPR and significantly attenuated hyperknesis, with no effect on spontaneous scratching or alloknesis. To investigate whether NK1R-expressing spinal neurons project in ascending somatosensory pathways, we performed a double-label study. The retrograde tracer, Fluorogold (FG), was injected into either the somatosensory thalamus or lateral parabrachial nucleus. In the upper cervical (C1-2) spinal cord, most neurons retrogradely labeled with FG were located in the dorsomedial aspect of the superficial dorsal horn. Of FG-labeled spinal neurons, 89% to 94% were double labeled for NK1R. These results indicate that NK1R-expressing spinal neurons play a major role in the expression of symptoms of chronic itch and give rise to ascending somatosensory projections. Gastrin-releasing peptide receptor-expressing spinal neurons contribute to hyperknesis but not to alloknesis or ongoing itch. NK1R-expressing spinal neurons represent a potential target to treat chronic itch.

In clinical trials, overexpression of neurokinin-1 receptors (NK1R) in gliomas has been exploited by intratumoral injection of its radiolabeled ligand, substance P (SP). However, despite proven NK1R expression, patients' response to the therapy was inhomogeneous. This study aims to identify the factors predicting response to NK1R-targeted glioma therapy, thereby allowing the discrimination between potential "responders" and "nonresponders" and thus a personalized therapeutic approach. Four widely used glioblastoma cell lines were examined concerning their RNA levels of full-length and truncated NK1R subtypes. Binding of SP to NK1R and internalization into glioma cells was studied by three different approaches using radiolabeled SP ((177)Lu-[DOTA, Thi(8), Met(O2)(11)]-SP), a fluorescence-labeled SP derivative (SP-FAM), and a toxin-SP conjugate (saporin-SP). While NK1R RNA was detected in all cases, receptor subtype analysis revealed impressive differences between the cell lines; LN319 exhibited the highest level of full-length NK1R RNA. Significant binding of SP conjugates to NK1R, cell internalization, and specific cell killing were only observed with the cell line LN319. Thus, different NK1R subtype profiles of glomerular basement membrane (GBM) cell lines appear to influence the binding of SP conjugates and their cell internalization properties. Both processes are crucial steps for NK1R-based targeted therapy. Pretherapeutic testing for NK1R subtype expression may therefore be advisable before initiation of this generally promising therapeutic modality.

The substance P neurokinin 1 receptor (NK1R) regulates motility, secretion, inflammation and pain in the intestine. The distribution of the NK1R is a key determinant of the functional effects of substance P in the gut. Information regarding the distribution of NK1R in subtypes of mouse enteric neurons is lacking and is the focus of the present study. NK1R immunoreactivity (NK1R-IR) is examined in whole-mount preparations of the mouse distal colon by indirect immunofluorescence and confocal microscopy. The distribution of NK1R-IR within key functional neuronal subclasses was determined by using established neurochemical markers. NK1R-IR was expressed by a subpopulation of myenteric and submucosal neurons; it was mainly detected in large multipolar myenteric neurons and was colocalized with calcitonin gene-related peptide, neurofilament M, choline acetyltransferase and calretinin. The remaining NK1R-immunoreactive neurons were positive for nitric oxide synthase. NK1R was expressed by most of the submucosal neurons and was exclusively co-expressed with vasoactive intestinal peptide, with no overlap with choline acetyltransferase. Treatment with substance P resulted in the concentration-dependent internalisation of NK1R from the cell surface into endosome-like structures. Myenteric NK1R was mainly expressed by intrinsic primary afferent neurons, with minor expression by descending interneurons and inhibitory motor neurons. Submucosal NK1R was restricted to non-cholinergic secretomotor neurons. These findings highlight key differences in the neuronal distribution of NK1R-IR between the mouse, rat and guinea-pig, with important implications for the functional role of NK1R in regulating intestinal motility and secretion.

Background: The tachykininergic neurotransmitters have been proved to be involved in the inflammatory progress and chronic pain in series of disease. The present study was undertaken to evaluate the levels of substance P (SP) and its receptors NK-1 receptor (NK-1R) in both serum and synovial tissues of hip joint from patients with different stages of DDH, and to detect the possible correlation of serum SP levels with pain sensation and dysfunction of the hip joint., Methods: SP levels in serum and synovial tissues from patients with DDH and DDH combined with osteoarthritis (DDH&OA) group were compared through immunohistochemistry (IHC), ELISA, and 2-step acetic acid extraction method respectively. Expression of NK-1R in synovial tissues was compared through IHC, quantitive Real-Time PCR (QRT-PCR) and Western-Blot. The severities of pain sensation and the functional activities of hip joint were assessed by Visual analogue scale (VAS) and Harris hip score (HHS). Correlations of serum SP levels with VAS, HHS and erythrocyte sedimentation rate (ESR) were evaluated respectively in these groups., Results: Significantly elevated serum SP levels were detected in group of DDH and DDH&OA compared to that in normal group. IHC, QRT-PCR as well as tissue Elisa showed that SP levels in synovial tissue of DDH&OA group is stronger than that in DDH group. Serum SP levels in each group have no gender differences. The enhanced SP levels in synovial tissue mainly came from the segregation of peripheral nerve endings. Serum SP correlated with VAS and HHS in patients with DDH&OA (Male + Female). Serum SP correlated with HHS in patients with DDH (Male). Serum SP levels also correlated with erythrocyte sedimentation rate (ESR) in patients with DDH&OA (Male + Female). Up-regulated expression of NK-1R was also observed in synovial tissue of patients with DDH&OA compared to patients with DDH, through western-blot, IHC, and QRT-PCR., Conclusions: These findings indicated that the increasing SP levels in serum and synovial tissues, observed from patients with DDH to patients with DDH&OA, might associate with the loss of function and chronic pain sensation in hip joint. SP along with its receptors NK-1R might be involved in the progression of DDH into DDH&OA. In the future, inhibitors of SP as well as NK-1R may represent a novel pharmacotherapy target for pain relieving, inflammation alleviating and joint degeneration delaying for patients with DDH.

Osteosarcoma is a highly malignant bone tumor in children and adolescents. Aprepitant is a selective high‑affinity antagonist of the human neurokinin‑1 (NK‑1) receptor (NK1R) with robust antitumor activity. No data exist on the presence of NK1R in osteosarcoma and whether this tumor responds to NK1R antagonists. Here, we analyzed the expression of NK1R in the human osteosarcoma cell line MG-63 with western blot analysis and PCR and found significant expression both at the protein and mRNA levels. We further studied the growth inhibitory capacity of aprepitant and other NK1R antagonists on MG-63 in vitro using an MTS cytotoxicity assay and DAPI staining. All antagonists induced tumor growth inhibition and apoptosis. Synergism was observed for the combination of L-733,060 with common cytostatic drugs in MG-63, but not in non-malignant HEK293 cells. Pretreatment of HEK293 with L-733,060 prior to exposure to cytostatic drugs partially protected HEK293 cells from inhibition by these drugs. Furthermore, nanomolar concentrations of substance P (SP), the natural ligand of the NK1R, increased the growth rate of MG‑63 cells and micromolar concentrations of aprepitant inhibited SP-induced growth in a dose‑dependent manner. In vivo, a xenograft for MG-63 was created in nude mice and treated with peritumoral s.c. injections of fosaprepitant, which resulted in a significant reduction of tumor volume. Collectively, we demonstrated for the first time that the NK1R is expressed in human osteosarcoma cell line MG‑63 and that this receptor can be targeted with NK1R antagonists both in vitro as well as in vivo.

Background and Objective: Although 5-fluorouracil (5-FU) is a widely used as chemotherapy agent, severe mucositis develops in approximately 80% of patients. 5-FU-induced small intestinal mucositis can cause nausea and vomiting. The current study was designed to investigate peripheral alterations due to the 5-FU-induced mucositis of neuronal and non-neuronal 5-HT3 and NK1 receptor expression by immunohistochemical analysis., Methods: 5-FU was administered by i.p. injection to C57BL/6 mice. After 4 days, segments of the jejunum were removed. The specimens were analyzed by immunohistochemistry, real-time PCR, and enzyme immunoassay., Results: The numbers of 5-HT3 receptor immunopositive cells and nerve fibers in mucosa were increased by 5-FU treatment. The 5-HT3 receptor immunopositive cell bodies were found only in jejunal submucosa and myenteric plexus in the 5-FU-treated mice. The numbers of NK1 receptor cells in mucosa and immunopositive expression of NK1 receptors in deep muscular plexus were dramatically increased in 5-FU-treated mice. Real-time PCR demonstrated that 5-FU treatment significantly increased mRNA levels of 5-HT3A, 5-HT3B, and NK1 receptors. The amounts of 5-HT and substance P increased after 5-FU treatment. The 5-HT3 or NK1 receptor immunopositive cells colocalized with both 5-HT and substance P. Furthermore, 5-HT3 and NK1 receptors colocalized with CD11b., Conclusions: The 5-HT3 and NK1 immunopositive macrophages and mucosal mast cells in lamina propria release 5-HT and substance P, which in turn activate their corresponding receptors on mucosal cells in autocrine and paracrine manners. It is assumed to result in the release of 5-HT and substance P in mucosa.

The tachykinin neuropeptide substance P (SP) has an important signaling role in both the nervous and the immune systems. Two naturally occurring variants of the neurokinin-1 receptor (NK1R) mediate the effects of SP, full-length receptor (NK1R-F) and a truncated form (NK1R-T) that lacks 96 amino acid residues at the C-terminus. We previously reported decreased expression of the NK1R-F in the CNS of HIV-positive individuals in comparison to HIV-negative control subjects. There were no differences in the expression of the NK1R-T in the same groups. In the current study, we quantified the expressions of SP precursor mRNA preprotachykinin (TAC1), NK1R (full and truncated forms), viral load (HIV-gag) and several proinflammatory and immune markers (CD4, CCR5, CXCR4, fractalkine, IL-6, IL-10, CCL2, CCL20 and CD163) in the frontal cortex of autopsied brains from HIV-1-positive individuals with or without HIV-associated neuropathology. The expressions of SP and, to lesser extent, NK1R-F were decreased while the expressions of CXCR4, CCR5 and CCL2 were increased in CNS of individuals with HIV-associated neuropathology. There was no change in HIV loads associated with neuropathology; however, we found a positive correlation between viral loads and the expression of haptoglobin-hemoglobin scavenger receptor CD163. An analysis of CSF from corresponding samples demonstrated an increase in proinflammatory markers (CCL2 MIP-1α and MIP-1β) associated with neuropathology. Although our data confirm the overall inflammatory nature of HIV-associated neuropathology, we observed a decrease in the expression of SP and NK1R-F, which is also associated with other forms of neuroinflammation., (© 2013 Elsevier B.V. All rights reserved.)

Background: Substance P is a sensory nerve neuropeptide located near coronary vessels in the heart. Therefore, substance P may be one of the first mediators released in the heart in response to hypertension, and can contribute to adverse myocardial remodeling via interactions with the neurokinin-1 receptor. We asked: 1) whether substance P promoted cardiac hypertrophy, including the expression of fetal genes known to be re-expressed during pathological hypertrophy; and 2) the extent to which substance P regulated collagen production and fibrosis., Methods and Results: Spontaneously hypertensive rats (SHR) were treated with the neurokinin-1 receptor antagonist L732138 (5mg/kg/d) from 8 to 24 weeks of age. Age-matched WKY served as controls. The gene encoding substance P, TAC1, was up-regulated as blood pressure increased in SHR. Fetal gene expression by cardiomyocytes was increased in SHR and was prevented by L732138. Cardiac fibrosis also occurred in the SHR and was prevented by L732138. Endothelin-1 was up-regulated in the SHR and this was prevented by L732138. In isolated cardiac fibroblasts, substance P transiently up-regulated several genes related to cell-cell adhesion, cell-matrix adhesion, and extracellular matrix regulation, however, no changes in fibroblast function were observed., Conclusions: Substance P activation of the neurokinin-1 receptor induced expression of fetal genes related to pathological hypertrophy in the hypertensive heart. Additionally, activation of the neurokinin-1 receptor was critical to the development of cardiac fibrosis. Since no functional changes were induced in isolated cardiac fibroblasts by substance P, we conclude that substance P mediates fibrosis via up-regulation of endothelin-1., (© 2013.)

It is well known that neurons in the rostral ventromedial medulla (RVM) are involved in descending modulation of nociceptive transmission in the spinal cord. It has been shown that activation of neurokinin-1 receptors (NK-1Rs) in the RVM, which are presumably located on pain facilitating ON cells, produces hyperalgesia whereas blockade of NK-1Rs attenuates hyperalgesia. To obtain a better understanding of the functions of NK-1R expressing neurons in the RVM, we selectively ablated these neurons by injecting the stable analog of substance P (SP), Sar(9),Met(O2)(11)-Substance P, conjugated to the ribosomal toxin saporin (SSP-SAP) into the RVM. Rats received injections of SSP-SAP (1 μM) or an equal volume of 1 μM of saporin conjugated to artificial peptide (Blank-SAP). Stereological analysis of NK-1R- and NeuN-labeled neurons in the RVM was determined 21-24 days after treatment. Withdrawal responses to mechanical and heat stimuli applied to the plantar hindpaw were determined 5-28 days after treatment. Withdrawal responses were also determined before and after intraplantar injection of capsaicin (acute hyperalgesia) or complete Freund's adjuvant (CFA) (prolonged hyperalgesia). The proportion of NK-1R-labeled neurons in the RVM was 8.8 ± 1.3% in naïve rats and 8.1 ± 0.8% in rats treated with Blank-SAP. However, injection of SSP-SAP into the RVM resulted in a 90% decrease in NK-1R-labeled neurons. SSP-SAP did not alter withdrawal responses to mechanical or heat stimuli under normal conditions, and did not alter analgesia produced by morphine administered into the RVM. In contrast, the duration of nocifensive behaviors produced by capsaicin and mechanical and heat hyperalgesia produced by capsaicin and CFA were decreased in rats pretreated with SSP-SAP as compared to those that received Blank-SAP. These data support our earlier studies using NK-1R antagonists in the RVM and demonstrate that RVM neurons that possess the NK-1R do not play a significant role in modulating acute pain or morphine analgesia, but rather are involved in pain facilitation and the development and maintenance of hyperalgesia., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Autonomic neurons innervate pancreatic islets of Langerhans and maintain blood glucose homeostasis by regulating hormone levels. We previously showed that cell adhesion molecule 1 (CADM1) mediated the attachment and interaction between nerves and aggregated pancreatic islet α cells. In this study, we cocultured αTC6 cells, a murine α cell line, with mouse superior cervical ganglion (SCG) neurons. The oscillation of intracellular Ca(2+) concentration ([Ca(2+)]i) was observed in 27% and 14% of αTC6 and CADM1-knockdown αTC6 cells (αTC6(siRNA-CADM1) cells) in aggregates, respectively, within 1min after specific SCG nerve stimulation with scorpion venom. In αTC6(siRNA-CADM1) cells, the responding rate during 3min after SCG nerve stimulation significantly increased compared with that within 1min, whereas the increase in the responding rate was not significantly different in αTC6 cells. This indicated that the response of αTC6 cells according to nerve stimulation occurred more rapidly and effectively than that of αTC6(siRNA-CADM1) cells, suggesting CADM1 involvement in promoting the interaction between nerves and α cells and among α cells. In addition, because we found that neurokinin (NK)-1 receptors, which are neuropeptide substance P receptors, were expressed to a similar extent by both cells, we investigated the effect of substance P on nerve-α cell interaction. Pretreatment with CP99,994 (0.1μg/ml), an NK-1 receptor antagonist, reduced the responding rate of both cells, suggesting that substance P released from stimulated neurites was a mediator to activate αTC6 cells. In addition, α cells that were attached to neurites in a CADM1-mediated manner appeared to respond effectively to neurite activation via substance P/NK-1 receptors., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Aims: Obesity and asthma are widely prevalent and associated disorders. Recent studies of our group revealed that Substance P (SP) is involved in pathophysiology of obese-asthma phenotype in mice through its selective NK1 receptor (NK1-R). Lymphangiogenesis is impaired in asthma and obesity, and SP activates contractile and inflammatory pathways in lymphatics. Our aim was to study whether NK1-R expression was involved in lymphangiogenesis on visceral (VAT) and subcutaneous (SAT) adipose tissues and in the lungs, in obese-allergen sensitized mice., Main Methods: Diet-induced obese and ovalbumin (OVA)-sensitized Balb/c mice were treated with a selective NK1-R antagonist (CJ 12,255, Pfizer Inc., USA) or placebo. Lymphatic structures (LYVE-1+) and NK1-R expression were analyzed by immunohistochemistry. A semi-quantitative score methodology was used for NK1-R expression., Key Findings: Obesity and allergen-sensitization together increased the number of LYVE-1+ lymphatics in VAT and decreased it in SAT and lungs. NK1-R was mainly expressed on adipocyte membranes of VAT, blood vessel areas of SAT, and in lung epithelium. Obesity and allergen-sensitization combined increased the expression of NK1-R in VAT, SAT and lungs. NK1-R antagonist treatment reversed the effects observed in lymphangiogenesis in those tissues., Significance: The obese-asthma phenotype in mice is accompanied by increased expression of NK1-R on adipose tissues and lung epithelium, reflecting that SP released during inflammation may act directly on these tissues. Blocking NK1-R affects lymphangiogenesis, implying a role of SP, with opposite physiological consequences in VAT, and in SAT and lungs. Our results provide a clue for a novel SP role in the obese-asthma phenotype., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Obesity, inflammation, and insulin resistance are closely linked. Substance P (SP), via its neurokinin 1 receptor (NK1R), mediates inflammatory and, possibly, neuroendocrine processes. We examined SP effects on lipid storage and cytokine production in 3T3-L1 adipocytes and adipose tissues. 3T3-L1 adipocytes and preadipocytes express NK1R, and 8 days of SP supplementation during differentiation to 3T3-L1 preadipocytes decreased lipid droplet accumulation. SP (10 nM, 24 h) increased lipolysis in primary adipocytes (138 ± 7%, P < 0.05) and reduced fatty acid uptake (-31 ± 7%, P < 0.05) and mRNA expression of the differentiation-related transcription factors peroxisome proliferator-activated receptor-γ type 2 (-64 ± 2%, P < 0.001) and CCAAT enhancer-binding protein (CEBP)-α (-65 ± 2%, P < 0.001) and the lipid storage genes fatty acid-binding protein type 4 (-59 ± 2%, P < 0.001) and diacylglycerol O-acyltransferase-1 (-45 ± 2%, P < 0.01) in 3T3-L1 adipocytes, while CD36, a fatty acid transporter (+82 ± 19%, P < 0.01), was augmented. SP increased secretion of complement C3 (148 ± 15%, P < 0.04), monocyte chemoattractant protein-1 (156 ± 16%, P < 0.03), and keratinocyte-derived chemokine (148 ± 18%, P = 0.045) in 3T3-L1 adipocytes and monocyte chemoattractant protein-1 (496 ± 142%, P < 0.02) and complement C3 (152 ± 25%, P < 0.04) in adipose tissue and primary adipocytes, respectively. These SP effects were accompanied by downregulation of insulin receptor substrate 1 (-82 ± 2%, P < 0.01) and GLUT4 (-76 ± 2%, P < 0.01) mRNA expression, and SP acutely blocked insulin-mediated stimulation of fatty acid uptake and Akt phosphorylation. Although adiponectin secretion was unchanged, mRNA expression was decreased (-86 ± 8%, P < 0.001). In humans, NK1R expression correlates positively with plasma insulin, fatty acid, and complement C3 and negatively with adiponectin, CEBPα, CEBPβ, and peroxisome proliferator-activated receptor-γ mRNA expression in omental, but not subcutaneous, adipose tissue. Our results suggest that, beyond its neuroendocrine and inflammatory effects, SP could also be involved in targeting adipose tissue and influencing insulin resistance.

Lesions that remove neurons expressing neurokinin-1 (NK1) receptors from the nucleus tractus solitarii (NTS) without removing catecholaminergic neurons lead to loss of baroreflexes, labile arterial pressure, myocardial lesions, and sudden death. Because destruction of NTS catecholaminergic neurons expressing tyrosine hydroxylase (TH) may also cause lability of arterial pressure and loss of baroreflexes, we sought to test the hypothesis that cardiac lesions associated with lability are not dependent on damage to neurons with NK1 receptors but would also occur when TH neurons in NTS are targeted. To rid the NTS of TH neurons we microinjected anti-dopamine β-hydroxylase conjugated to saporin (anti-DBH-SAP, 42 ng/200 nl) into the NTS. After injection of the toxin unilaterally, immunofluorescent staining confirmed that anti-DBH-SAP decreased the number of neurons and fibers that contain TH and DBH in the injected side of the NTS while sparing neuronal elements expressing NK1 receptors. Bilateral injections in eight rats led to significant lability of arterial pressure. For example, on day 8 standard deviation of mean arterial pressure was 16.8 ± 2.5 mmHg when compared with a standard deviation of 7.83 ± 0.33 mmHg in six rats in which phosphate buffered saline (PBS) had been injected bilaterally. Two rats died suddenly at 5 and 8 days after anti-DBH-SAP injection. Seven-treated animals demonstrated microscopic myocardial necrosis as reported in animals with lesions of NTS neurons expressing NK1 receptors. Thus, cardiac and cardiovascular effects of lesions directed toward catecholamine neurons of the NTS are similar to those following damage directed toward NK1 receptor-containing neurons.

The substance P/NK1 receptor system plays an important role in the regulation of stress and emotional responding and as such had been implicated in the pathophysiology of anxiety and depression. The present study investigated whether alterations in the substance P/NK1 receptor system in brain areas which regulate emotional responding accompany the depressive behavioural phenotype observed in the olfactory bulbectomised (OB) mouse. The effect of NK1 receptor deletion on behavioural responding and monoamine levels in discrete brain regions of the OB model, were also examined. Substance P levels in the frontal cortex and NK1 receptor expression in the amygdala and hippocampus were enhanced following olfactory bulbectomy. Although NK1 receptor knockout (NK1-/-) mice did not exhibit altered behavioural responding in the open field test, noradrenaline levels were enhanced in the frontal cortex, amygdala and hippocampus, as were serotonin levels in the frontal cortex. Locomotor activity and exploratory behaviour were enhanced in wild type OB mice, indicative of a depressive-like phenotype, an effect attenuated in NK1-/- mice. Bulbectomy induced a decrease in noradrenaline and 5-HIAA in the frontal cortex and an increase in serotonin in the amygdala, effects attenuated in OB NK1-/- mice. The present studies indicate that alterations in substance P/NK1 receptor system underlie, at least in part, the behavioural and monoaminergic changes in this animal model of depression., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Injection into the nucleus tractus solitarii (NTS) of toxins that target substance P (SP) receptors ablates neurons that express neurokinin-1 (NK1) receptors, attenuates baroreflexes, and results in increased lability of arterial pressure. We and others have shown that the toxin leads to loss of neurons containing SP receptors and loss of GABAergic neurons in the NTS; but given that neither type neuron is thought to be integral to baroreflex transmission in NTS, mechanisms responsible for the cardiovascular changes remained unclear. Because NK1 receptors colocalize with N-methyl-d-aspartate (NMDA) receptors and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in NTS and because glutamate transmission may be integral to baroreflex transmission in the NTS we hypothesized that the toxic lesions may interrupt mechanisms for glutamate transmission. Interruption of those mechanisms could be responsible for the cardiovascular effects. We tested the hypothesis by performing fluorescent immunohistochemistry, confocal microscopy and image analysis after injecting stabilized SP-SAP (SSP-SAP) unilaterally into the NTS. We assessed changes in immunoreactivity (IR) of NMDA receptor subunit 1 (NMDAR1), AMPA receptor subunit 2 (GluR2), and 3 types of vesicular glutamate transporters (VGluT) as well as IR of gamma-aminobutyric acid receptors type b (GABAb), neuronal nitric oxide synthase (nNOS), tyrosine hydroxylase (TH), and protein gene product 9.5 (PGP 9.5), a neuronal marker, in the NTS. When compared to that of the same section of the un-injected NTS, IR decreased significantly in the injected side for NMDAR1 (p<0.01), GluR2 (p<0.01), VGluT3 (p<0.01), GABAb (p<0.001), and PGP9.5 (p<0.001). In contrast, IR for VGluT1 (p<0.001), VGluT2 (p<0.001), nNOS (p<0.001), and TH (p<0.001) increased significantly. We conclude that pathologic effects following ablation of neurons with NK1 receptors in NTS may result from interruption of neurotransmission through other neurochemical systems associated with NK1 receptors-containing neurons., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Caveolin (Cav)-1 is an integral membrane protein of caveolae playing a crucial role in various signal transduction pathways. Caveolae represent the sites for calcium entry and storage especially in smooth muscle cells (SMC) and interstitial cells of Cajal (ICC). Cav-1(-/-) mice lack caveolae and show abnormalities in pacing and contractile activity of the small intestine. Presently, we investigated, by transmission electron microscopy (TEM) and immunohistochemistry, whether the absence of Cav-1 in Cav-1(-/-) mouse small intestine affects ICC, SMC and neuronal morphology, the expression of NK1 and NK2 receptors, and of Ano1 (also called Dog1 or TMEM16A), an essential molecule for slow wave activity in gastrointestinal muscles. ICC were also labelled with c-Kit and tachykinergic neurons with Substance P (SP). In Cav-1(-/-) mice: (i) ICC were Ano1-negative but maintained c-Kit expression, (ii) NK1 and NK2 receptor immunoreactivity was more intense and, in the SMC, mainly intracytoplasmatic, (iii) SP-immunoreactivity was significantly reduced. Under TEM: (i) ICC, SMC and telocytes lacked typical caveolae but had few and large flask-shaped vesicles we called large-sized caveolae; (ii) SMC and ICC contained an extraordinary high number of mitochondria, (iii) neurons were unchanged. To maintain intestinal motility, loss of caveolae and reduced calcium availability in Cav-1-knockout mice seem to be balanced by a highly increased number of mitochondria in ICC and SMC. Loss of Ano-1 expression, decrease of SP content and consequently overexpression of NK receptors suggest that all these molecules are Cav-1-associated proteins., (© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Substance P (SP), a neuropeptide member of the tachykinin (TK) family, has a functional role both in physiological and pathological conditions, including Amyotrophic Lateral Sclerosis disease. One hypothesis of the selective motor neuron death in ALS involves the excitatory neurotransmitter glutamate, because these neurons are extremely susceptible to excessive stimulation of AMPA receptors. It has been reported that SP exerts its action against a variety of insults including excitotoxicity, and that altered levels of SP have been observed in the cerebrospinal fluid (CSF) of patients with ALS. Here we have analyzed the interaction between SP and AMPA receptor functionality, both in Control cortical neurons in culture and in those obtained from a genetic mouse model of ALS (G93A). Our studies demonstrate that SP reduces the kainate-activated currents in Control and G93A neurons and that this reduction is significantly higher in the mutated neurons. SP effect is mediated by its receptor NK1 because GR 82334 (5 μM), a NK1 competitive antagonist, is able to suppress the current reduction. Analysis of miniature excitatory postsynaptic currents (mEPSCs) in Control and G93A neurons indicates that SP (200 nM) is able to significantly decrease the mEPSC amplitudes in G93A neurons, whereas it is ineffective on Control mEPSCs. Western blotting experiments in cultures and cortical tissues show a higher NK1 expression level in G93A mice compared to that of Control. This is also confirmed by immunocytochemistry experiments in cultured neurons. In addition, the amount of GluR1 subunit AMPA receptors is not modified following SP exposure, indicating a non internalization of the AMPA receptors. Finally, toxicity experiments have revealed that SP is able to rescue G93A cortical cells whereas it is ineffective on those of Control. These findings provide the first evidence of SP having a physiological and protective role in the G93A mouse model of ALS, and may suggest the possible use of SP as a clinical therapeutic treatment., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Substance P (SP) is involved in the pathophysiology of acute pancreatitis (AP) via binding to its high-affinity receptor, neurokinin-1-receptor (NK1R). An up-regulation of SP and NK1R expression was observed in experimental AP and in caerulein-stimulated pancreatic acinar cells. However, the mechanisms that lead to this up-regulation are not fully understood. In this study, we showed the role of protein kinase C (PKC) in caerulein-induced SP and NK1R production in isolated mouse pancreatic acinar cells. Caerulein (10(-7) M) stimulation rapidly activated the conventional PKC-α and novel PKC-δ as observed by the phosphorylation of these molecules. Pre-treatment of pancreatic acinar cells with Gö6976 (1-10 nM) and rottlerin (1-10 μM) inhibited PKC-α and PKC-δ phosphorylation, respectively, but not the other way round. At these concentrations used, PKC-α and PKC-δ inhibition reversed the caerulein-induced up-regulation of SP and NK1R, indicating an important role of PKCs in the modulation of SP and NK1R expression. Further experiments looking into signalling mechanisms showed that treatment of pancreatic acinar cells with both Gö6976 and rottlerin inhibited the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). Inhibition of PKC-α or PKC-δ also affected caerulein-induced transcription factor activation, as represented by nuclear factor-κB and AP-1 DNA-binding activity. The findings in this study suggested that PKC is upstream of the mitogen-activated protein kinases and transcription factors, which then lead to the up-regulation of SP/NK1R expression in caerulein-treated mouse pancreatic acinar cells., (© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Central sensitization in inflammatory pain conditions results in behavioral mechanical hypersensitivity. Specifically, C-fiber-driven spinal hyperexcitability enables A fibers to gain access to specific spinal circuitry, via heterosynaptic facilitatory mechanisms, to mediate mechanical hypersensitivity. However, the precise circuitry engaged is not known. Lamina I neurokinin 1 (NK1) receptor expressing (NK1R(+)) dorsal horn neurons, many of which are projection neurons, are required for the development of this hypersensitivity and are therefore likely to be a component of this circuitry. To investigate, whole-cell patch-clamp recordings were made from lamina I NK1R(+) neurons in the spinal cord slice preparation with attached dorsal root, obtained from rats with or without complete Freund's adjuvant (CFA) hindpaw inflammation. EPSCs were recorded in response to electrical stimulation of the dorsal root. Control neurons predominantly received monosynaptic C-fiber input (69%) with a smaller proportion receiving monosynaptic Aδ-fiber input (28%). In contrast, CFA inflammation significantly increased the incidence (by twofold) and magnitude (by 75% in a subset) of monosynaptic Aδ-fiber but not monosynaptic C-fiber-evoked responses. Aβ-fiber input to lamina I NK1R(+) neurons was minimal, polysynaptic in nature, and unaltered by CFA inflammation. Additional examination of control neurons revealed that a proportion received silent monosynaptic Aδ-fiber input, suggesting that these may provide the substrate for the novel Aδ inputs observed in CFA inflammation. This inflammation induced unmasking and strengthening of monosynaptic Aδ drive to lamina I NK1R(+) neurons may contribute to the heterosynaptic facilitatory mechanisms underlying mechanical hyperalgesia in inflammatory pain.

The analgesic properties of the opium poppy Papever somniferum were first mentioned by Hippocrates around 400 BC, and opioid analgesics remain the mainstay of pain management today. These drugs can cause the serious side-effect of respiratory depression that can be lethal with overdose, however the critical brain sites and neurochemical identity of the neurons mediating this depression are unknown. By locally manipulating neurotransmission in the adult rat, we identify the critical site of the medulla, the preBötzinger complex, that mediates opioid-induced respiratory depression in vivo. Here we show that opioids at the preBötzinger complex cause respiratory depression or fatal apnea, with anesthesia and deep-sleep being particularly vulnerable states for opioid-induced respiratory depression. Importantly, we establish that the preBötzinger complex is fully responsible for respiratory rate suppression following systemic administration of opioid analgesics. The site in the medulla most sensitive to opioids corresponds to a region expressing neurokinin-1 receptors, and we show in rhythmically active brainstem section in vitro that neurokinin-1 receptor-expressing preBötzinger complex neurons are selectively inhibited by opioids. In summary, neurokinin-1 receptor-expressing preBötzinger complex neurons constitute the critical site mediating opioid-induced respiratory rate depression, and the key therapeutic target for its prevention or reversal.

Psychological stress and estrogen status are risk factors to develop painful temporomandibular joint disorders (TMJD); however, the neural basis for this relationship is not known. This study tested the hypothesis that repeated forced swim stress and estradiol treatment alter the phosphorylation of cAMP responsive element-binding protein (pCREB) in trigeminal subnucleus caudalis (Vc), the initial site of sensory input from the TMJ. Ovariectomized female rats were given low or high dose estradiol and subjected to repeated forced swim stress for 3 days and on day 4 an intra-TMJ injection of mustard oil or vehicle was given. Forced swim alone increased the number of pCREB-positive neurons, independent of estradiol treatment or TMJ stimulation, in superficial and deep laminae of Vc. Forced swim also increased the number of Fos-positive neurons in superficial laminae and neurokinin-1 receptor mRNA in whole dorsal Vc, independent of estradiol treatment. These results indicated that persistent psychophysical stress alone was sufficient to increase the expression of pCREB and downstream regulated genes associated with enhanced excitability in the caudal medullary dorsal horn, a brainstem region thought to be critical for TMJD pain., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Palonosetron is the only 5-HT(3) receptor antagonist approved for the treatment of delayed chemotherapy-induced nausea and vomiting (CINV) in moderately emetogenic chemotherapy. Accumulating evidence suggests that substance P (SP), the endogenous ligand acting preferentially on neurokinin-1 (NK-1) receptors, not serotonin (5-HT), is the dominant mediator of delayed emesis. However, palonosetron does not bind to the NK-1 receptor. Recent data have revealed cross-talk between the NK-1 and 5HT(3) receptor signaling pathways; we postulated that if palonosetron differentially inhibited NK-1/5-HT(3) cross-talk, it could help explain its efficacy profile in delayed emesis. Consequently, we evaluated the effect of palonosetron, granisetron, and ondansetron on SP-induced responses in vitro and in vivo. NG108-15 cells were preincubated with palonosetron, granisetron, or ondansetron; antagonists were removed and the effect on serotonin enhancement of SP-induced calcium release was measured. In the absence of antagonist, serotonin enhanced SP-induced calcium-ion release. After preincubation with palonosetron, but not ondansetron or granisetron, the serotonin enhancement of the SP response was inhibited. Rats were treated with cisplatin and either palonosetron, granisetron, or ondansetron. At various times after dosing, single neuronal recordings from nodose ganglia were collected after stimulation with SP; nodose ganglia neuronal responses to SP were enhanced when the animals were pretreated with cisplatin. Palonosetron, but not ondansetron or granisetron, dose-dependently inhibited the cisplatin-induced SP enhancement. The results are consistent with previous data showing that palonosetron exhibits distinct pharmacology versus the older 5-HT(3) receptor antagonists and provide a rationale for the efficacy observed with palonosetron in delayed CINV in the clinic.

Lamina I of the spinal cord contains many projection neurons that express the neurokinin 1 receptor (NK1r). It has been reported that these cells can undergo long-term potentiation (LTP), which may result from insertion of AMPA-type glutamate receptors (AMPArs) containing GluA1 or GluA4 subunits. We therefore investigated synaptic AMPAr expression on these cells with immunocytochemistry following antigen-retrieval. We also examined their density of glutamatergic input (by analysing AMPAr synaptic puncta and contacts from glutamatergic boutons), and phosphorylation of extracellular signal-regulated kinases (pERKs) following noxious stimulation. Our results indicate that there are two populations of NK1r-expressing projection neurons: large GluA4(+)/GluA1(-) cells with a high density of glutamatergic input and small GluA1(+)/GluA4(-) cells with a much lower input density. Results from pERK experiments suggested that the two groups may not differ in the types of noxious stimulus that activate them. Glutamatergic synapses on distal dendrites of the large cells were significantly longer than those on proximal dendrites, which presumably compensates for the greater attenuation of distally-generated excitatory postsynaptic currents (EPSCs). Both types of cell received contacts from peptidergic primary afferents, however, on the large cells these appeared to constitute over half of the glutamatergic synapses, and were often associated with elongated AMPAr puncta. This suggests that these afferents, which probably contain substance P, provide a powerful, secure synaptic input to large NK1r-expressing projection neurons. These results demonstrate the importance of GluA4-containing AMPArs in nociceptive transmission and raise the possibility that different forms of LTP in lamina I projection neurons may be related to differential expression of GluA1/GluA4., (Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

In the brain, the stress system plays an important role in motivating continued alcohol use and relapse. The neuropeptide substance P and the neurokinin-1 receptor (NK1R) are involved in the stress response and drug reward systems. Recent findings have shown that the binding of ligands to NK1Rs decreases the self-administration of alcohol in mice. We examined the effect of an artificial microRNA (amiRNA) on the functional expression of NK1R in mouse brains. Lentiviruses expressing either an amiRNA targeting the NK1R (amiNK1R) or a negative control amiRNA (amiNC) were injected into mouse brains. Four weeks after amiRNA injection, we found that amiNK1R decreased the voluntary alcohol consumption compared to mice injected with amiNC. We also observed that NK1R expression was reduced in the hippocampus. RNA interference is an effective approach to regulate the expression of specific behavior-related genes. Our results support the potential use of amiRNA as a therapeutic agent for the treatment of alcohol dependence., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

The neuropeptide substance P (SP) has emerged to be an important proinflammatory mediator in acute pancreatitis (AP). The presence of substance P and its receptor, neurokinin-1 receptor (NK1R) has been shown in the pancreas and the pancreatic acinar cells. In this study, we investigated the unexplored mechanisms that mediate SP and NK1R expression using an in vitro AP model. Pancreatic acinar cells were obtained from pancreas of male Swiss mice. Isolated cells were treated with caerulein to mimic secretagogue pancreatitis. A concentration-dependent study that subjected the cells to 60 min of stimulation by caerulein showed that SP and the transcript from its gene preprotachykinin-A (PPT-A), and NK1R were up-regulated at a supraphysiological concentration of 10(-7) M. A concentration-dependent study on intracellular kinases, extracellular signal-regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK) and also transcription factors nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) showed that they were activated when the caerulein concentration was 10(-7) M. Inhibition of JNK reversed the up-regulation of PPT-A, SP, and NK1R. However, inhibition of ERK1/2 reversed the up-regulation of NK1R but not of PPT-A and SP. Furthermore, we found that specific ERK1/2 and JNK inhibitors reduce NF-kappaB and AP-1 activity. Taken together, our results suggest that supraphysiological concentrations of caerulein up-regulate the expression of SP and NK1R in pancreatic acinar cells, and the signaling molecules that are involved in this up-regulation include ERK1/2, JNK, NF-kappaB, and AP-1.

Lamina I of the spinal dorsal horn contains neurons that project to various brain regions, and approximately 80% of these projection cells express the neurokinin 1 receptor (NK1r), the main receptor for substance P. Two populations of NK1r-immunoreactive neurons have been identified in lamina I: small weakly immunoreactive cells and large cells with strong immunolabelling [Cheunsuang O and Morris R (2000) Neuroscience 97:335-345]. The main aim of this study was to test the hypothesis that the large cells are projection neurons and that the small cells are interneurons. Projection neurons were identified by injection of tracers into the caudal ventrolateral medulla and lateral parabrachial area, and this was combined with immunostaining for NK1r. We found a bimodal size distribution for NK1r-immunoreactive neurons. The small cells (with somatic cross-sectional areas <200 microm(2)) showed weak immunoreactivity, while immunostaining intensity was variable among the large cells. Virtually all (99%) of the immunoreactive cells with soma areas >200 microm(2) were retrogradely labelled, while only 10% of retrogradely labelled cells were smaller than this. Soma sizes of retrogradely labelled neurons that lacked NK1r did not differ from those of NK1r-expressing projection neurons. It has been suggested that a population of small pyramidal projection neurons that lack NK1r may correspond to cells activated by innocuous cooling, and we therefore assessed the morphology of retrogradely labelled cells that were not NK1r-immunoreactive. Fifteen percent of these were pyramidal, but these did not differ in size from pyramidal NK1r-immunoreactive projection neurons. These results confirm that large NK1r-immunoreactive lamina I neurons are projection cells, and suggest that the small cells are interneurons. Since almost all of the NK1r-immunoreactive cells with soma size >200 microm(2) were retrogradely labelled, cells of this type can be identified as projection cells in anatomical studies.

The objectives of this study were to investigate the presence and distribution of substance P and neurokinin 1 receptor in oral premalignant epithelium and their relation with the presence of dysplasia, and to analyze whether the expression of substance P can be considered an early oncogenic event in oral carcinogenesis. substance P and neurokinin 1 receptor expression was immunohistochemically studied in 83 oral carcinomas and adjacent non-tumor epithelia. The presence and degree of epithelial dysplasia was assessed according to WHO criteria. The nuclear, cytoplasmic, and membrane expression of substance P and the cytoplasmic and membrane expression of neurokinin 1 receptor were assessed in tumor and adjacent non-tumor epithelium. Nuclear and cytoplasmic expression of substance P in non-tumor epithelium was significantly associated with the presence of epithelial dysplasia (p<0.001) and carcinoma in situ (p=0.021). Nuclear, cytoplasmic, and membrane expressions of substance P in non-tumor epithelium were significantly (p<0.001) associated with its expression in the corresponding tumor. These findings suggest that substance P plays a role in early oral carcinogenesis by promoting the proliferation and growth of premalignant fields.

Musculoskeletal injury and inflammation is associated with performance of repetitive and forceful tasks. In this study, we examined the effects of performing a voluntary, highly repetitive, negligible force (HRNF) reaching task on spinal cord neurochemicals involved in nociception. To our knowledge, no other laboratories are examining spinal cord nociceptive neurochemicals in response to repetitive motion-induced injury and inflammation. The purpose of this study was to extend our earlier findings related to central neurotransmitters from a low demand task to a higher demand task. Specifically, this study determined immunoreactivity of a peptidergic pro-nociceptive transmitter (substance P) and one of its receptors, neurokinin-1 (NK-1) receptor, in spinal cord dorsal horns in rats performing a HRNF reaching task for 6-10 weeks. The relationship of these spinal cord changes with the number of TNFalpha immunopositive cells in flexor forelimb muscles and with previously observed forearm grip strength changes from these same rats were examined. Performance of the HRNF task resulted in significantly increased substance P and NK-1 receptor immunoreactivity in the superficial lamina of spinal cord dorsal horns at 6 and 10 weeks compared to trained controls (p<0.01). The increased substance P and NK-1 receptor immmunoreactivity were positively correlated with declines in forearm grip strength, an assay of movement-related hyperalgesia (r=0.70, p<0.01 and r=0.64, p<0.05, respectively). The increased substance P and NK-1 receptor immmunoreactivity were also positively correlated with increased TNF immunopositive cells in forelimb flexor muscles (r=0.85, p<0.001 and r=0.88, p<0.001, respectively). Thus, our highly repetitive task leads to increased spinal cord pro-nociceptive neurochemicals that are most likely directed by forelimb muscle inflammation and pain.

Tachykinins are important neurotransmitters regulating intestinal motility. Slow transit constipation (STC) represents an extreme colonic dysmotility with unknown etiology that predominantly affects women. We examined whether the tachykinin system is involved in the pathogenesis of STC. Isolated sigmoid colon circular muscle from female STC and control patients was studied using functional and quantitative reverse transcriptase-polymerase chain reaction methods. A possible alteration of neurotransmission was investigated by electrical field stimulation (EFS) and ganglionic stimulation by dimethylphenylpiperazinium (DMPP). Substance P (SP)-mediated contractions in circular muscle strips were significantly diminished in STC compared with age-matched control (P < 0.001). In contrast, contractile responses to neurokinin A, the selective tachykinin NK(2) receptor agonist, [Lys(5),MeLeu(9),Nle(10)]NKA(4-10), and acetylcholine were unaltered in STC. The reduced responses to SP in STC were fully restored by indomethacin, partially reversed by tetrodotoxin (TTX), but unaffected by atropine or hexamethonium. The restoration by indomethacin was blocked by the NK(1) receptor antagonist CP99994 [(2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine] and TTX. In STC colonic muscle, there was a significant increase of NK(1) receptor mRNA expression, but no difference in NK(2) mRNA level. DMPP generated biphasic responses, relaxation at lower and contraction at higher concentrations. Although the responses to DMPP were similar in STC and control, an altered contractile pattern in response to EFS was observed in STC circular muscle. In conclusion, we postulate that the diminished contractile response to SP in STC is due to an increased release of inhibitory prostaglandins through activation of up-regulated NK(1) receptors. Our results also indicate some malfunction of the enteric nervous system in STC.

Background and Purpose: The metalloendopeptidase endothelin-converting enzyme 1 (ECE-1) is prominently expressed in the endothelium where it converts big endothelin to endothelin-1, a vasoconstrictor peptide. Although ECE-1 is found in endosomes in endothelial cells, the role of endosomal ECE-1 is unclear. ECE-1 degrades the pro-inflammatory neuropeptide substance P (SP) in endosomes to promote recycling and re-sensitization of its neurokinin 1 (NK(1)) receptor. We investigated whether ECE-1 regulates NK(1) receptor re-sensitization and the pro-inflammatory effects of SP in the endothelium., Experimental Approach: We examined ECE-1 expression, SP trafficking and NK(1) receptor re-sensitization in human microvascular endothelial cells (HMEC-1), and investigated re-sensitization of SP-induced plasma extravasation in rats., Key Results: HMEC-1 expressed all four ECE-1 isoforms (a-d), and fluorescent SP trafficked to early endosomes containing ECE-1b/d. The ECE-1 inhibitor SM-19712 prevented re-sensitization of SP-induced Ca2+ signals in HMEC-1 cells. Immunoreactive ECE-1 and NK(1) receptors co-localized in microvascular endothelial cells in the rat. SP-induced extravasation of Evans blue in the urinary bladder, skin and ears of the rat desensitized when the interval between two SP injections was 10 min, and re-sensitized after 480 min. SM-19712 inhibited this re-sensitization., Conclusions and Implications: By degrading endocytosed SP, ECE-1 promotes the recycling and re-sensitization of NK(1) receptors in endothelial cells, and thereby induces re-sensitization of the pro-inflammatory effects of SP. Thus, ECE-1 inhibitors may ameliorate the pro-inflammatory actions of SP.

Neurogenic inflammation, an important component of many disease states, is mediated by the release of neuropeptides from sensory nerves. To date, it has been possible to inhibit neurogenic inflammation using neuropeptide receptor blockers or by prevention of neuropeptide release. In the current edition of the British Journal of Pharmacology, Cattaruzza and co-workers discuss a novel way of blocking the action of neuropeptides. They have shown that the re-sensitization of the substance P neurokinin-1 receptor and the substance P-induced pro-inflammatory effects are mediated by the enzyme, endothelin-converting enzyme 1 (ECE-1). Therein, they showed that ECE-1 inhibition could prevent the re-sensitization process. This is exciting progress in our understanding of neurogenic inflammation, but it remains to be seen how inhibition of receptor recycling via ECE-1 blockade will affect other inflammatory pathways.

Neurogenic inflammation describes the local release of neuropeptides, notably substance P (SP), from afferent neurons and might play a role in the pathogenesis of pulpal disease. The fibroblast is the most numerous cell type in the dental pulp, and recent work has suggested that it is involved in the inflammatory response. Primary pulp fibroblast cell populations were isolated by enzymatic digestion. Whole pulp tissue was obtained from freshly extracted sound (n = 35) and carious (n = 39) teeth. Expression of SP and neurokinin-1 receptor (NK-1) mRNA by pulp fibroblasts was determined by reverse transcriptase polymerase chain reaction (RT-PCR). SP was expressed by pulpal fibroblasts at both mRNA and protein levels. In addition, NK-1 mRNA and protein expression was detected in fibroblast cultures by RT-PCR and Western blotting, respectively. SP levels, determined by radioimmunoassay, were significantly greater (P < .05) in carious compared with sound teeth. These findings suggest that pulp fibroblasts play a role in neurogenic inflammation in pulpal disease.

Activity in descending systems from the brainstem modulates nociceptive transmission through the dorsal horn. Intrathecal injection of the neurotoxin saporin conjugated to SP (SP-SAP) into the lumbar spinal cord results in the selective ablation of NK(1) receptor expressing (NK(1)+ve) neurones in the superficial dorsal horn (lamina I/III). Loss of these NK(1)+ve neurones attenuates excitability of deep dorsal horn neurones due to a disruption of both intrinsic spinal circuits and a spino-bulbo-spinal loop, which activates a descending excitatory drive, mediated through spinal 5HT(3) receptors. Descending inhibitory pathways also modulate spinal activity and hence control the level of nociceptive transmission relayed to higher centres. To ascertain the spinal origins of the major descending noradrenergic inhibitory pathway we studied the effects of a selective alpha2-adrenoceptor antagonist, atipamezole, on neuronal activity in animals pre-treated with SP-SAP. Intrathecal application of atipamezole dose dependently facilitated the mechanically evoked neuronal responses of deep dorsal horn neurones to low intensity von Frey hairs (5-15 g) and noxious thermal (45-50 degrees C) evoked responses in SAP control animals indicating a physiological alpha2-adrenoceptor control. This facilitatory effect of atipamezole was lost in the SP-SAP treated group. These data suggest that activity within noradrenergic pathways have a dependence on dorsal horn NK(1)+ve cells. Further, noradrenergic descending inhibition may in part be driven by lamina I/III (NK(1)+ve) cells, and mediated via spinal alpha2-adrenoceptor activation. Since the same neuronal population drives descending facilitation and inhibition, the reduced excitability of lamina V/VI WDR neurones seen after loss of these NK(1)+ve neurones indicates a dominant role of descending facilitation.