NK1 Receptor mRNA Research Articles

IL-4 and IFN-gamma up-regulate substance P receptor expression in murine peritoneal macrophages.

While the ability of macrophages to express authentic substance P receptors (i.e., NK-1 receptors) has been inferred from radioreceptor binding assays and functional assays and, most recently, by identification of NK-1 receptor mRNA expression, we know little about NK-1 expression at the protein level or what host factors might up-regulate expression of this receptor. In the present study we demonstrate that the cytokines IL-4 and IFN-gamma can increase the expression of NK-1 receptors on murine peritoneal macrophages. Specifically, we show that IL-4 and IFN-gamma can elicit increases in the level of mRNA encoding the NK-1 receptor by up to 12- and 13-fold, respectively. Furthermore, these cytokines can significantly increase the expression of the NK-1 receptor protein as measured by Western blot and FACS analysis using specific Abs developed in our laboratory. In addition, we have demonstrated the ability of both IL-4 and IFN-gamma to enhance the ability of macrophages to bind substance P as measured by radiolabeled binding assay. The observation that the level of expression of this receptor protein can be enhanced by cytokines that promote either cell-mediated (Th1) or humoral (Th2) immune responses supports the idea that this receptor can be induced during either type of immune response. As such, these results may point to a more ubiquitous role for substance P in the generation of optimal immune responses than previously appreciated.

Tachykinin receptor and neutral endopeptidase gene expression in the rat uterus: characterization and regulation in response to ovarian steroid treatment.

Tachykinin neuropeptides, such as substance P, are localized to a population of sensory fibers that innervate the mammalian female reproductive tract. In the present study, we have characterized tachykinin NK1 receptor (NK1R), NK2 receptor (NK2R), and NK3 receptor (NK3R) gene expression by semiquantitative RT-PCR in uteri from ovariectomized rats and studied their regulation in response to 17beta-estradiol (E2), progesterone (P4), or a combination of both. In addition, we analyzed the expression and regulation of the neutral endopeptidase 24.11 (NEP), the most important enzyme involved in tachykinin degradation in the rat uterus. In uteri from control (olive oil-treated) rats, RT-PCR assays revealed single bands corresponding to the expected product sizes encoding complementary DNA for NK1R (232 bp), NK2R (491 bp), NK3R (325 bp), and NEP (221 bp). The identity of the amplified fragments was confirmed by DNA sequence analysis. Compared with control rats, NK1R messenger RNA (mRNA) was increased by 2-fold in uteri from rats treated with E2, was decreased by 3.3-fold in rats treated with P4, and was decreased by 1.8-fold in rats treated with both E2 and P4. Uterine NK2R mRNA levels were not altered by any steroid treatment. E2 treatment decreased by 15-fold NK3R mRNA. P4 was without effect if administered alone and did not influence the E2-induced decrease in NK3R mRNA. NEP mRNA levels were about 4-fold lower in E2-treated than in P4-treated rats. Functional studies were carried out in uteri from E2- or P4-treated ovariectomized rats to characterize the contractile response evoked by the selective tachykinin receptor agonists [Sar9Met(O2)11]substance P (NK1R selective), [Nle10]NKA-(4-10) (NK2R selective), and [MePhe7]NKB (NK3R selective) in the presence of the NEP inhibitor phosphoramidon (1 microM). A marked correlation was observed between the magnitude of the contractile response to each agonist and the level of expression determined by RT-PCR for each tachykinin receptor. The present findings show that tachykinin NK1R, NK2R, NK3R, and NEP are expressed in the rat uterus and that ovarian steroids differentially regulate their expression.

Functional neurokinin NK-1 receptor expression in rat peritoneal mast cells.

Recently, Ogawa et al. [17] reported that the peritoneal mast cells (PMCs) of rats can release histamine by substance P (SP) in a receptor-dependent manner. In the present study, we confirmed and extended their findings. PMCs were isolated from six strains of rats. In some experiments, peritoneal cells in the non-MC fraction were used. PMCs were incubated with SP, neurokinin (NK) receptor agonists or antagonists, and histamine content in the supernatant was measured. In the binding assay, PMCs were incubated with [125I]BH-SP together with SP or NK receptor antagonists. NK-1 receptor mRNA was detected using a reverse transcription-polymerase chain reaction (RT-PCR) assay. PMCs from Slc: Wistar and F344/NSlc were highly sensitive to SP, leading to histamine release, whereas those from Slc:SD and three other strains were not. PMCs from Slc:Wistar and F344/NSlc also released histamine in the presence of an NK-1 agonist. The histamine release induced by SP and the NK-1 agonist was inhibited by the NK-1 receptor antagonists, FK888 and CP-99,994. [125I]BH-SP binding experiments revealed that PMCs from Slc:Wistar rats possessed a single high affinity binding site for SP and that the binding was blocked by NK-1 receptor antagonists. Peritoneal cells in the non-MC fraction exhibited no appreciable binding. In the RT-PCR assay, expression of NK-1 receptor mRNA was evident in Slc:Wistar PMCs, but not in the non-MC fraction from Slc:Wistar or Slc: SD PMCs. These data demonstrate the existence of functional NK-1 receptors on freshly isolated PMCs in at least some strains of rats.

Expression and presence of septal neurokinin-2 receptors controlling hippocampal acetylcholine release during sensory stimulation in rat.

We examined the expression and presence of NK2 receptors in the septal area of rat brain, and investigated their functional role in the regulation of the septohippocampal cholinergic system. Using reverse transcription-polymerase chain reaction (RT-PCR) analysis, we showed the presence of NK2 receptor mRNA expression in the septal area, and detected septal NK2 binding sites by using a fluorescent-tagged neurokinin A (NKA) derivative. In vivo microdialysis was employed to explore the functional role of NK2 receptors in the release of hippocampal acetylcholine evoked by tactile stimulation in freely moving rats. Two sessions of stroking of the neck and back of the rat for 30 min, at 90 min intervals, produced a marked and reproducible increase in hippocampal acetylcholine release. This effect was dose-dependently prevented by intraperitoneal administration of the two selective non-peptide tachykinin NK2 receptor antagonists SR144190 (0.03-0.3 mg/kg, i.p.) and SR48968 (0.3 and 1 mg/kg, i.p.), but not by the inactive enantiomer of SR48968 (SR48965, 1 mg/kg) nor by the two non-peptide NK1 receptor antagonists SR140333 (3 mg/kg, i.p.) and GR205171 (1 mg/kg, i.p.). Furthermore, the intraseptal application of SR144190 (10(-8) M) reduced the sensory response. Finally, intraseptal perfusion of neurokinin A (0.01-10 microM) in anaesthetized rats produced a concentration-dependent increase in hippocampal acetylcholine release. The response to neurokinin A (0.1 microM) was prevented by SR144190 (0.03-0.3 mg/kg, i.p.) and SR48968 (0.3-1 mg/kg, i.p.). In conclusion, this study provides direct evidence for the role of endogenous NKA/substance P, through the activation of NK2 receptors, in regulating the septohippocampal cholinergic function.

Small sensory neurons in the rat dorsal root ganglia express functional NK-1 tachykinin receptor.

The tachykinins substance P (SP) and neurokinin A, released by the C-type primary afferent fibre terminals of the small dorsal root ganglion (DRG) neurons, play important roles in spinal nociception. By means of non-radioactive in situ hybridization and whole-cell recording, we showed that the small rat DRG neurons also express the NK-1 tachykinin receptor. In situ hybridization demonstrated that the positive neurons in rat DRG sections were mainly small cells (85.9%) with diameters less than 25 microm. The remaining positive neurons (14.1%) were cells with medium diameters between 26 and 40 microm. No positive large neurons (diameters > 40 microm) were observed. Expression in small DRG neurons (diameter < 21 microm) was confirmed by in situ hybridization of isolated cells, which were demonstrated to express NK-1 receptor mRNA at a very high frequency (> 90% of small DRG neurons) and therefore were subjected to whole-cell recording. In 57 of 61 cells recorded, SP or the selective NK-1 receptor agonist [Sar9, Met(O2)11]SP (Sar-SP, 1 or 2 microM) produced a delayed vibrating inward current (50-300 nA) with a long duration of 0.5-2 h. These currents were blocked by co-application of the NK-1 receptor antagonist L-668, 169 (1 microM), but were not affected by the NK-2 antagonist L-659, 877 (2 microM). Both current-clamp recording and cell-attached single-channel recording demonstrated that the long-lasting response was due to the opening of a channel with an inward current. Employment of non-Ca2+ and Ca2+ + choline solutions revealed that this channel might be a Ca2+-permeable, non-selective cation channel. The prolonged NK-1 tachykinin response exhibited extreme desensitization. This work suggests that presynaptic NK-1 autoreceptors may be present on the primary afferent terminals in the spinal cord, where they could contribute to the chronic pain and hyperalgesia.

Glucocorticoids reduce tachykinin NK2 receptor expression in bovine tracheal smooth muscle.

Neurokinin A is not only a potent bronchoconstrictor, but also has immuno-modulatory effects in animals and man, mediated via tachykinin NK2 receptors. We have examined the effect of the glucocorticoid, dexamethasone, on tachykinin NK2 receptor mRNA and the number of tachykinin NK2 receptors in bovine tracheal smooth muscle in vitro by Northern blot analysis using a human tachykinin NK2 receptor cDNA probe and receptor binding assay using [3H]SR48968 [(S)-N-methyl-N[4-acetylamino-4-phenylpiperidino-2(3,4-dichlorophenyl) butyl]benzamide]. Tachykinin NK2 receptor mRNA showed a time-dependent suppression (62% reduction after 6 h at 10(-7) M of dexamethasone), as well as a concentration-dependent suppression after the incubation with dexamethasone (IC50 = 1.3 x 10(-8) M). This suppression was abolished by the glucocorticoid receptor antagonist, mifepristone (RU38486), indicating that dexamethasone acts via the glucocorticoid receptor. It was also abolished by the protein synthesis inhibitor, cycloheximide (10 microg/ml), indicating that new protein synthesis is required on this suppression. Using the RNA polymerase inhibitor actinomycin D (5 microg/ml), we showed that the stability of tachykinin NK2 receptor mRNA was not affected by dexamethasone (t1/2 = 5 h). Nuclear run-on assays revealed a 51% reduction in the rate of tachykinin NK2 receptor gene transcription after treatment with dexamethasone for 6 h. Radioligand binding assay using an selective tachykinin NK2 receptor antagonist, [3H]SR48968 showed a significant decrease in the number of receptor binding sites after 16 h (Bmax = 262 +/- 23 versus 213 +/- 13 fmol/mg protein for vehicle and dexamethasone treatment respectively, P < 0.05), with no significant change at the earlier time points. These results suggest that glucocorticoids act on glucocorticoid receptors to decrease tachykinin NK2 receptor expression by decreasing the rate of tachykinin NK2 receptor gene transcription.

Expression of Substance P (NK1) Receptor mRNA in Human Nose

In airway tissues, it has been suggested that substance P is the transmitter of afferent sensory nerves which respond to various irritants and may be involved in airway allergic reactions. Three classes of tachykinin receptor are currently recognized, denoted as NK1, NK2 and NK3, which exhibit preferential affinity for substance P, neurokinin A and neurokinin B, respectively. We used molecular probes to study the gene expression and distribution of NK1 receptor in human nose. Total RNA was isolated from human nasal mucosa and NK1 receptor mRNA was detected in these tissues by using reverse transcription and polymerase chain reaction (RT-PCR). For in situ hybridization study of human nasal mucosa, we utilized the PCR directly to incorporate a T7 RNA polymerase promoter sequence onto the NK1 receptor cDNA, and these PCR products were used as the DNA templates for producing digoxigenin-labelled anti-sense and sense RNA probes. These studies revealed that NK1 receptor mRNA was expressed in submucosal glands and airway epithelium. The results may have an important clinical implication, and also promote further investigation of gene regulation of NK1 receptor in health and disease.

Mouse bone marrow-derived mast cells acquire responsiveness to substance P after co-culture with 3T3 fibroblasts in the presence of stem cell factor

ABSTRACT Connective tissue-type mast cells degranulate in response to a neurogenic peptide, substance P , whereas bone marrow-derived mast cells (BMMC) do not respond to this stimulant when prepared with a combination of interleukin-3 and interleukin-4. In the present study we demonstrated that BMMC obtained from three different strains of mice, NC, BALB/c and C57BL/6, which are immature mast cells low in histamine content and unresponsive to substance F) increased 10 to 100-fold in their histamine content and acquired responsiveness to substance P after co-culture with NIH/3T3 fibroblasts in the presence of 10 ng/mL of stem cell factor (SCF). This change was observed after 1 week of co-culture and increased over a 3 week period, whereas 3T3 fibroblasts or SCF (100 ng/mL) alone was insufficient to duplicate this phenotypic change in BMMC. It is suggested that the response to substance P of mast cells is not through NK-1 receptors but rather through a different mechanism, since the reverse transcriptase-mediated polymerase chain reaction technique failed to show expression of NK-1 receptor mRNA in BMMC after co- culture as well as before co-culture.

O-269 - Single-cell RT-PCR analysis of NK1 receptor mRNA in primary sensory neurons of the mouse

O-269 - Single-cell RT-PCR analysis of NK1 receptor mRNA in primary sensory neurons of the mouse

2009 5-hydroxytryptamine release-mediated mechanisms of the central fatigue: Effect of enhanced tryptophan in the extrasynaptosomes

We previously reported that motion sickness was prevented in rats with amygdala lesion and that provocative motion stimuli increased the number of Fos-positive neurons in the amygdala, suggesting that the amygdala is one of the neural substrates involved in the development of motion sickness. NK-1 receptors in the brain stem and amygdala are thought to play an important role in emesis and affective disorders, respectively. In the present study, to elucidate a role of substance P neuronal system and NK-1 receptors in the brain stem and amygdala in the development of motion sickness, we measured changes in gene expression of NK-1 receptors and preprotachykinin, a precursor of substance P, using quantitative real-time PCR methods in solitary tract nucleus and amygdala in rats after provocative motion stimuli induced by 2G hypergravity load. Effects of systemic administration of CP-99,994, an antagonist for NK-1 receptors, on hypergravity-induced motion sickness were also examined using pica behavior, eating non-nutritive substances such as kaolin, as an index of motion sickness in rats. Hypergravity-induced motion sickness was inhibited by CP-99,994 with a dose-dependent and enantioselective manner. Preprotachykinin mRNA expression was increased in basolateral nucleus of amygdala and solitary tract nucleus after hypergravity load for 3 h, whereas NK-1 receptor mRNA expression was not changed by hypergravity in amygdala and solitary tract nucleus. Present results suggest that 2G hypergravity load activated the substance P neuronal system in amygdala as well as in the brain stem and this activation would be related to the development of motion sickness.

Expression of tachykinin NK1 receptor mRNA in dorsal root ganglia of the mouse

We examined whether mRNA coding for tachykinin NK1 receptor is expressed in the dorsal root ganglion (DRG) of the mouse, using reverse transcription-polymerase chain reaction (RT-PCR). Both the RT-PCR of the total RNA from the DRGs using four pairs of primers and the digestion of these products with the restriction enzymes gave bands with the predicted length. Further amplification (nested PCR) of part of one PCR product also gave a band with the predicted length. Southern blot hybridization of RT-PCR products of total RNA from the DRG and several CNS regions revealed that the expression level of NK1 receptor mRNA in the DRG was similar to the cerebellum and less than the olfactory bulb, cerebral cortex, medulla oblongata and spinal cord. The present results suggest that NK1 receptor mRNA is expressed in the mouse DRG, although the level is relatively low.

Expression of NK-1 receptor mRNA in murine T lymphocytes.

Substance P (SP), one of the best characterized neurogenic mediators of immune hyperactivity, stimulates the production of a variety of cytokines, and acts as a general proinflammatory agent inducing proliferation and activation of immune cells Neurokinin 1 receptor (NK-1R), a G-protein-coupled receptor with high affinity for SP, has been cloned from nonlymphoid tissues. Although previous binding studies indicated the presence of high affinity SP receptors on immune cells, recent studies questioned the existence of specific NK-1R on lymphocytes and monocytes. In this study we investigate the expression of the NK-1R gene in murine T lymphocytes and T cell lines. The expression of NK-1R mRNA was determined by reverse transcription polymerase chain reaction (RT-PCR), with two separate sets of specific primers, and by nuclease protection assays. The NK-1R nature of the lymphocytic amplified fragments was confirmed by Southern blots with specific murine brain NK-1R probes. Both T cells lines (EL-4.IL-2 and LBRM-T6G) which were previously shown to respond to SP by increased IL-2 production express NK-1R mRNA constitutively. Normal spleen cells and purified CD4+ T lymphocytes which also responded to SP by increased IL-2 production express NK-1R mRNA. In addition to CD4+ T cells, NK-1R message is also expressed in murine splenic B lymphocytes. Sequencing of the RT-PCR amplified fragments from EL-4.IL-2 cells, purified CD4+ T lymphocytes, and murine brain showed complete identity to the published murine NK-1R sequence. This study indicates that murine CD4+ T lymphocytes possess the molecular template for the production of NK-1R. Together with previous binding studies, and with pharmacological studies regarding the effect of SP and of selective NK-1R antagonists on cytokine production, the present study supports the hypothesis that the response of CD4+ T cells to SP and related tachykinins is mediated through specific NK-1 receptors.

Substance P (NK1)- and neurokinin A (NK2)-receptor gene expression in inflammatory airway diseases.

The tachykinin neuropeptides substance P and neurokinin (NK) A have been postulated to participate in the inflammatory reaction in airways of smokers and asthmatics. We have examined the hypothesis that the expression of one or more of the three cloned tachykinin receptors (NK1, NK2, and NK3) is increased in inflammatory airway disorders, which could result in augmentation of the effect of released tachykinin neuropeptides. NK1 receptor and NK2 receptor but not NK3-receptor mRNA were detected by ribonuclease protection assay in RNA from both cartilaginous and membranous bronchi and subpleural lung. In lung samples containing membranous airways, NK2-receptor mRNA expression was increased fourfold in asthmatics compared with nonsmoking controls, whereas NK1-receptor mRNA levels were similar in the two groups. NK1- and NK2-receptor mRNA expression was increased twofold in smokers without airflow obstruction compared with nonsmokers, whereas NK1-receptor mRNA expression was significantly lower in patients with chronic obstructive pulmonary disease compared with smoking controls. In situ hybridization indicated NK1-receptor mRNA was expressed in submucosal glands and airway epithelial cells, whereas NK2-receptor and NK3-receptor mRNA were not detected. These observations have implications for the pathophysiology and treatment of both asthma and tobacco smoke-induced airway inflammation.

Alterations in neurokinin 1 receptor gene expression in models of pain and inflammation.

Substance P and the related tachykinin peptides are involved in inflammatory processes and in the transmission of sensory nociceptive information. In this article we review the evidence implicating substance P and the neurokinin 1 (NK1) receptor in arthritic disease. We also provide preliminary evidence demonstrating that cultured synoviocytes from a patient with rheumatoid arthritis express NK1 receptor mRNA that can be downregulated by tumor necrosis factor alpha, whereas synoviocytes from a normal patient do not express detectable NK1 receptor mRNA or protein. Data are also presented summarizing recent studies on nociception-induced increases in sensory ganglia of levels of mRNA encoding substance P and increases in dorsal horn NK1 receptor mRNA levels. Morphine pretreatment blocked the increases in dorsal horn NK1 receptor mRNA levels but did not block the nociception-induced substance P encoding mRNA levels in sensory ganglia. These results are discussed with reference to mechanisms that may regulate P turnover and NK1 receptor sensitivity in models of pain and inflammation.

Functional expression of the tachykinin NK1 receptor by floor plate cells in the embryonic rat spinal cord and brainstem.

1. The floor plate is a ventral mid-line structure that plays a pivotal role in the organization of the developing vertebrate central nervous system. Previous studies have demonstrated that the floor plate may provide signals that induce neuronal differentiation and guide axons; however, it is not known whether the floor plate can itself respond to signals that derive from surrounding tissue. 2. The peptide substance P is one of the first transmitters to be expressed in the developing spinal cord. To determine whether the floor plate may respond to substance P we have examined the expression of the principal substance P receptor (the tachykinin NK1 receptor) by floor plate cells of the rat embryonic spinal cord using immunocytochemistry, in situ hybridization and fura-2 calcium imaging. 3. Immunocytochemistry demonstrated selective expression of the NK1 receptor by cells at the ventral mid-line of the spinal cord. Double immunofluorescence labelling with the specific floor plate marker FP3 indicated that NK1 receptor expression is confined to cells in the lateral region of the floor plate. 4. In order to confirm the specificity of the NK1 receptor immunoreactivity we performed in situ hybridization histochemistry using antisense cRNA probes directed against the NK1 receptor. In situ hybridization demonstrated selective expression of NK1 receptor mRNA by floor plate cells. 5. The ontogeny of NK1 receptor protein and mRNA expression in the floor plate was defined. NK1 receptor expression occurred in a rostrocaudal progression that begins at embryonic day 10-11 (E10-E11) and is complete by E12-E14. The restriction of NK1 receptor expression to the lateral part of the floor plate was conserved throughout embryonic development. 6. NK1 receptor signalling was assessed by monitoring substance P-evoked changes in the intracellular concentration of calcium ions ([Ca2+]i) of acutely dissociated cells from the floor plate region. Application of substance P (5 nM) elevated [Ca2+]i in 10% of cells examined. 7. Selective neurokinin agonists were used to identify the receptor subtype involved in the substance P-evoked elevation of [Ca2+]i. Acetyl-[Arg6,Sar9,Met(O2)11]-substance P(6-11) (5 nM) and [Sar9,Met(O2)11]-substance P (5 nM), two highly selective NK1 receptor agonists, both elevated [Ca2+]i in floor plate cells that responded to substance P. [beta-Ala8]-neurokinin A(4-10) (50 nM) and senktide (50 nM), selective agonists respectively of NK2 and NK3 receptors, had no effect on [Ca2+]i.

Molecular evidence that granuloma T lymphocytes in murine schistosomiasis mansoni express an authentic substance P (NK-1) receptor.

Murine Schistosomiasis mansoni is a parasitic disease in which granulomas develop around the schistosome ova that lodge in the liver and intestines of the host. The granuloma eosinophils make substance P (SP), a cytokine with immunoregulatory properties. Within the granuloma SP can modulate IFN-gamma production through interaction with a substance P-like receptor. SP belongs to a family of hormones called tachykinins. Three mammalian tachykinins are SP, neurokinin A (substance K), and neurokinin B (neuromedin K). In humans and rats, there are at least three distinct tachykinin receptors designated NK-1, NK-2, and NK-3. The NK-1 receptor binds only SP with high affinity. Using reverse transcription-PCR, cDNA cloning, and sequence analysis, we showed that granulomas isolated from the liver of infected mice express an authentic SP (NK-1) receptor but have no detectable neurokinin A (NK-2) and neurokinin B (NK-3) receptor mRNA, as determined by PCR. CD4+ granuloma T lymphocytes, purified by FACS, express NK-1 receptor mRNA. Normal liver devoid of granulomas exhibited none of the three tachykinin receptor subclasses.

Localization of NK1 receptor mRNA in rat spinal cord: Selective upregulation in unilateral peripheral inflammation

Localization of NK1 receptor mRNA in rat spinal cord: Selective upregulation in unilateral peripheral inflammation

Recent Results in Cancer Research

Tachykinins such as substance P (SP) may be involved in the pathogenesis of inflammatory airway diseases such as asthma. This study investigated the presence of SP and its receptor in the differentiated macrophage-like U-937 cell line and in macrophages from sputum induced in healthy subjects (n=8). In situ hybridization with digoxigenin-labelled sense and antisense complementary ribonucleic acid (cRNA) probes was used to determine the expression of SP and its receptor (neurokinin (NK)1 receptor). SP-immunoreactive material was detected using a rabbit anti-SP antiserum and the alkaline phosphatase anti-alkaline phosphatase technique. Beta-preprotachykinin (PPT)-I messenger ribonucleic acid (mRNA) encoding SP, was detected using in situ hybridization in differentiated U-937 cells as well as in CD45+ human leukocyte antigen (HLA) DR+ sputum macrophages. The expression of the beta-PPT-I mRNA was increased in lipopolysaccharide (LPS)-stimulated U-937 cells. SP-immunoreactive material was found in differentiated U-937 cells and in CD68+ sputum macrophages. NK1 receptor mRNA was detected in differentiated U-937 cells and sputum macrophages. Incubation of U-937 cells with SP considerably increased the expression of NK1 receptor mRNA. This study demonstrates that human monocytes/macrophages express substance P and that this expression is upregulated by lipopolysacharide. Human monocytes/macrophages also express neurokinin1 receptor messenger ribonucleic acid, suggesting an autocrine effect of substance P on these cells.