Presence Of Substance P Research Articles

Substance P promotes immunotherapy efficacy for airway allergy

BackgroundAllergen-specific immunotherapy (AIT) has been employed in the treatment of allergic diseases for many years. However, the effectiveness of AIT requires improvement. Substance P (SP) can interact with immune cells, modulate immune cell activity, and regulate immune reaction. The purpose of this study is to use SP as an immune regulator to enhance the therapeutic efficacy of AIT. MethodsAn established mouse model of the airway allergy disorder (AAD) was employed with ovalbumin as a specific antigen. The AAD response was evaluated through established procedures. AAD mice were treated with AIT employing SP as an immune regulator. Dendritic cells were isolated from the airway tissues by magnetic cell sorting, and were analyzed by RNA sequencing (RNAseq). ResultsWe observed that after sensitization with ovalbumin, mice exhibited AAD-like symptoms, serum specific IgE, and Th2 polarization. The presence of SP in the course of sensitization prevented the development of AAD. Treating mice with SP by nasal instillations induced IL-10, but not TGF-β, in dendritic cells of the airway tissues. The most differentially expressed genes (DEG) in the dendritic cells were those related to the IL-10 expression, including Il10, Tac1r, and Mtor. The gene ontology analysis showed that these DEGs mainly mapped to the tachykinin-PI3K-AKT-mTOR pathway. The addition of SP substantially enhanced the therapeutic efficacy of AIT for AAD by inducing antigen specific type 1 regulatory T cells (Tr1 cells). ConclusionActing as an immune regulator, SP promotes the therapeutic efficacy for AAD by inducing antigen specific Tr1 cells in the airway tissues.

Replacement of Lost Substance P Reduces Fibrosis in the Diabetic Heart by Preventing Adverse Fibroblast and Macrophage Phenotype Changes.

Reduced levels of the sensory nerve neuropeptide substance P (SP) have been reported in the diabetic rat heart, the consequence being a loss of cardioprotection in response to ischemic post-conditioning. We considered whether this loss of SP also predisposes the heart to non-ischemic diabetic cardiomyopathy in the form of fibrosis and hypertrophy. We report that diabetic Leprdb/db mice have reduced serum SP and that administration of exogenous replacement SP ameliorated cardiac fibrosis. Cardiac hypertrophy did not occur in Leprdb/db mice. Cardiac fibroblasts exposed to high glucose converted to a myofibroblast phenotype and produced excess extracellular matrix proteins; this was prevented by the presence of SP in the culture media. Cardiac fibroblasts exposed to high glucose produced increased amounts of the receptor for advanced glycation end products, reactive oxygen species and inflammatory cytokines, all of which were prevented by SP. Cultured macrophages assumed an M1 pro-inflammatory phenotype in response to high glucose as indicated by increased TNF-α, CCL2, and IL-6. SP promoted a shift to the reparative M2 macrophage phenotype characterized by arginase-1 and IL-10. Leprdb/db mice showed increased left ventricular M1 phenotype macrophages and an increase in the M1/M2 ratio. Replacement SP in Leprdb/db mice restored a favorable M1 to M2 balance. Together these findings indicate that a loss of SP predisposes the diabetic heart to developing fibrosis. The anti-fibrotic actions of replacement SP involve direct effects on cardiac fibroblasts and macrophages to oppose adverse phenotype changes. This study identifies the potential of replacement SP to treat diabetic cardiomyopathy.

Substance P and Hemokinin 1 in Nasal Lavage Fluid of Patients with Chronic Sinusitis and Nasal Polyposis

This pilot study was undertaken to isolate and quantify substance P (SP) and hemokinin 1 (HK-1) in the nasal lavage fluid of patients with chronic rhinosinusitis with nasal polyps to better elucidate the pathophysiology underlying this inflammatory process, which remains poorly understood. Mucus samples were collected from this introductory cohort of 10 patients diagnosed with chronic rhinosinusitis with nasal polyps at Tufts Medical Center (Boston, Massachusetts). Relative levels of SP and HK-1 were measured with enzyme-linked immunosorbent assay methods. Both inflammatory neuropeptides were found in detectable and comparable amounts in patient samples and in concentrations up to 100-fold those established in past literature. The presence of SP and HK-1 necessitates further investigation into their role in nasal polyposis and the potentiation of the chronic inflammation inherent to chronic rhinosinusitis. Downregulating these peptides could therefore provide novel treatment targets to manage this disease process.

Substance P Reshapes Inspiratory and Postinspiratory Activity Generated in Horizontal Brainstem Slices

The triple oscillator hypothesis posits that breathing is generated by three rhythmogenic microcircuits contained within the ventrolateral medulla. The preBötzinger Complex (preBötC) generates the inexorable inspiratory phase of breathing, whereas the conditional postinspiratory and active expiratory phases of breathing are generated by the Postinspiratory Complex (PiCo) and lateral parafacial respiratory group (PFL), respectively. Neuromodulators including norepinephrine, opioids, and somatostatin are known to differentially modulate the activity of each rhythmic circuit, likely contributing to the independence of each breathing phase and the flexibility of the respiratory rhythm. However, whether the functional boundaries of each of these microcircuits are static or can change dynamically based on the neuromodulatory state of the network is unknown. The neuromodulator Substance P (SP) facilitates breathing frequency in vivo and also in medullary slices that isolate the preBötC in vitro. Using a neonatal mouse horizontal brainstem slice preparation that preserves the rostrocaudal extent of the ventral medulla, we tested the hypothesis that SP differentially modulates bursting activity in the preBötC and PiCo and explored how SP may reshape the distribution of inspiratory activity within the wider medullary network. Rhythmic bursting activity was simultaneously recorded from the preBötC and PiCo (n=5). As expected, recordings from the PiCo, located ~500μm rostral to the preBötC, revealed a slow postinspiratory rhythm with robust bursts occurring after only ~1/12 preBötC bursts under control conditions. Following introduction of 0.5–1.0μM SP into the bath superfusion solution, PiCo bursts decreased in amplitude (~40%) and accelerated such that they occurred 1:1 with preBötC bursts without changing the time interval between peak preBötC and PiCo activity. In some PiCo recordings, we noted a small amount of inspiratory activity synchronized with preBötC bursts that was eliminated in the presence of SP. To test whether SP alters the spatial distribution of inspiratory activity along the ventral medulla, we simultaneously recorded inspiratory population activity generated at the preBötC and 400–600μm rostral (n=8). Following application of 1μM SP frequency was increased (~90%) and inspiratory burst amplitude was reduced by ~20% at the preBötC, whereas burst amplitude decreased by ~50% in rostral recordings. Based on these preliminary experiments, we hypothesize that SP reshapes breathing activity generated within the medulla by facilitating postinspiratory activity and reducing the extent of inspiratory activity rostral of the preBötC to shrink the size of the active inspiratory network. Differential neuromodulatory properties and dynamic functional boundaries of the rhythmogenic circuits that generate breathing may contribute to the remarkable flexibility and robustness of this vital physiological behavior.Support or Funding InformationNIH F32 HL134207NIH R01 HL126523This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Substance P Attenuates Hypoxia/Reoxygenation-Induced Apoptosis via the Akt Signalling Pathway and the NK1-Receptor in H9C2Cells

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

Immunolocalization of Substance P and NK‐1 Receptor in ADIPOSE Stem Cells

Substance P (SP) is a neuropeptide belonging to the thachykinin peptide family. SP, after binding to its receptor, the neurokinin 1 receptor (NK1R), controls several transcription factors such as NF-κB, hypoxia inducible factor (HIF-1α), c-myc, c-fos, c-jun, and AP-1. SP and NK1R have a widespread distribution in both the central and peripheral nervous systems. They are also present in cells not belonging to the nervous system (immune cells, placenta, etc.). SP is located in all body fluids, that is, blood, cerebrospinal fluid, etc., making it ubiquitous throughout the human body. SP and NK1R genes are expressed in the stem cell line TF-1 and in primary stem cells derived from human placental cord blood. However, to our knowledge, the presence of SP and the NK1R receptor in adipose stem cells (ADSC) is unknown. We demonstrated by immunofluorescence the localization of SP and NK1R in human and rat ADSC. SP and NK1R are located in both the cytoplasm and the nucleus of these cells. The NK1R is higher in the nucleus than in the cytoplasm of ADSCs. By Western blot we demonstrated the presence of different isoforms of NK1R that have different subcellular locations in the ADSC. SP induces proliferation and mitogenesis through NK1R in ADSCs. These findings reported here for the first time suggest an important role for a SP/NK1R system, either as genetic and/or epigenetic factor, in both the cytoplasm and nucleus functions of the ADSCs. J. Cell. Biochem. 118: 4686-4696, 2017. © 2017 Wiley Periodicals, Inc.

Increased nuclear localization of substance P in human gastric tumor cells

Gastric cancer (GC) is an aggressive disease that remains the fourth most common type of cancer and is the second leading cause of cancer-related death worldwide. Treatment of advanced or metastatic GC has seen little progress and median overall survival in this group remains <1 year. It is urgent to investigate new mechanisms to understand GC progression. It is known that substance P (SP), after binding to the neurokinin-1 (NK-1) receptor, elicits GC proliferation; that GC cells and samples express NK-1 receptors; that NK-1 receptor antagonists, in a concentration dependent manner, inhibit the proliferation of GC cells and that these cells die by apoptosis. However, the presence of SP in GC and normal gastric cells is unknown. In order to know more on the involvement of the SP/NK-1 receptor system in GC, we studied in thirty human GC and normal gastric samples the immunolocalization of SP after using an immunohistochemical technique. SP was observed in the cytoplasm and in the nucleus of GC and normal gastric cells. The nuclear expression of SP was higher in GC cells than in normal cells. No significant difference was observed when the cytoplasmatic expression of SP in normal and GC cells was compared. The findings suggest that SP plays an important role in both nuclear function and GC.

Expression of Tachykinins and Tachykinin Receptors and Interaction with Kisspeptin in Human Granulosa and Cumulus Cells.

The neurokinin B/NK3 receptor (NK3R) and kisspeptin/kisspeptin receptor (KISS1R), two systems which are essential for reproduction, are coexpressed in human mural granulosa (MGC) and cumulus cells (CCs). However, little is known about the presence of other members of the tachykinin family in the human ovary. In the present study, we analyzed the expression of substance P (SP), hemokinin-1 (HK-1), NK1 receptor (NK1R), and NK2 receptor (NK2R) in MGCs and CCs collected from preovulatory follicles of oocyte donors at the time of oocyte retrieval. RT-PCR, quantitative RT-PCR, immunocytochemistry, and Western blotting were used to investigate the patterns of expression of tachykinin and tachykinin receptor mRNAs and proteins and the possible interaction between the tachykinin family and kisspeptin. Intracellular free Ca(2+) levels ([Ca(2+)]i) in MGCs after exposure to SP or kisspeptin in the presence of SP were also measured. We found that SP, HK-1, the truncated NK1R isoform NK1R-Tr, and NK2R were all expressed in MGCs and CCs. NK1R-Tr mRNA and NK2R mRNA and protein levels were higher in MGCs than in CCs from the same patients. Treatment of cells with kisspeptin modulated the expression of HK-1, NK3R, and KISS1R mRNAs, whereas treatment with SP regulated kisspeptin mRNA levels and reduced the [Ca(2+)]i response produced by kisspeptin. These data demonstrate that the whole tachykinin system is expressed and acts in coordination with kisspeptin to regulate granulosa cell function in the human ovary.

Corneal epithelial and neuronal interactions: Role in wound healing

Impaired corneal innervation and sensitivity are the main causes of corneal neurotrophic keratopathy which simultaneously also leads to poor epithelial wound healing. Restoration of the diminished communication between the corneal epithelium and trigeminal nerve is indispensable for the proper functioning of the epithelium. The present study aims to investigate corneal epithelial and trigeminal neuron interactions to shed light on corneal wound healing during neurotrophic keratopathy. Mouse trigeminal neurons and corneal epithelial cells were cultured according to standard methods. To study the effect of corneal epithelial cells on trigeminal neurons as well as the effect of trigeminal neurons on corneal epithelial cells during wound healing, conditioned media from the cultures of pure trigeminal neurons (CNM) and corneal epithelial cells (CEM) were collected freshly and applied on the other cell type. Neurite outgrowth assay and RT-PCR analysis using primers specific for substance P (SP), Map1a, Map1b were performed on trigeminal neurons in the presence of CEM. We observed an increase in the neurite outgrowth in the presence of CEM and also in co-culture with corneal epithelial cells. Increase in the expression of SP mRNA and a decrease in the expression of Map1b mRNA was observed in the presence of CEM. We also observed the presence of epithelial-to-mesenchymal transition (EMT)-like phenomenon during wound healing using a scratch assay in primary corneal epithelial cultures. This system was further employed to study the effect of CNM on corneal epithelial cells in the context of wound healing to find the effect of trigeminal neurons on epithelial cells. RT-PCR analysis of Pax6 expression in corneal epithelial cell cultures with scratch served as a positive control. Further, we also show the expression of bone morphogenetic protein 7 (BMP7) mRNA in corneal epithelial cells which is decreased gradually along with Pax6 mRNA when cultured together in the presence of CNM. The expression and down regulation of BMP7 in the presence of CNM was further confirmed at the protein level by western blotting. From this study it seems that the epithelial and neuronal interactions in the cornea may contribute to the corneal innervation as well as recovery of corneal epithelial cells during injury. Appraising the differences in the expression of various signalling molecules during EMT of epithelial cells in the presence of SP and BMP7 gives an insight into the detailed dissection of the involved signalling pathways to develop future therapeutics.

Substance P Immunoreactivity Exhibits Frequent Colocalization with Kisspeptin and Neurokinin B in the Human Infundibular Region

Neurons synthesizing neurokinin B (NKB) and kisspeptin (KP) in the hypothalamic arcuate nucleus represent important upstream regulators of pulsatile gonadotropin-releasing hormone (GnRH) neurosecretion. In search of neuropeptides co-expressed in analogous neurons of the human infundibular nucleus (Inf), we have carried out immunohistochemical studies of the tachykinin peptide Substance P (SP) in autopsy samples from men (21-78 years) and postmenopausal (53-83 years) women. Significantly higher numbers of SP-immunoreactive (IR) neurons and darker labeling were observed in the Inf of postmenopausal women than in age-matched men. Triple-immunofluorescent studies localized SP immunoreactivity to considerable subsets of KP-IR and NKB-IR axons and perikarya in the infundibular region. In postmenopausal women, 25.1% of NKB-IR and 30.6% of KP-IR perikarya contained SP and 16.5% of all immunolabeled cell bodies were triple-labeled. Triple-, double- and single-labeled SP-IR axons innervated densely the portal capillaries of the infundibular stalk. In quadruple-labeled sections, these axons formed occasional contacts with GnRH-IR axons. Presence of SP in NKB and KP neurons increases the functional complexity of the putative pulse generator network. First, it is possible that SP modulates the effects of KP and NKB in axo-somatic and axo-dendritic afferents to GnRH neurons. Intrinsic SP may also affect the activity and/or neuropeptide release of NKB and KP neurons via autocrine/paracrine actions. In the infundibular stalk, SP may influence the KP and NKB secretory output via additional autocrine/paracrine mechanisms or regulate GnRH neurosecretion directly. Finally, possible co-release of SP with KP and NKB into the portal circulation could underlie further actions on adenohypophysial gonadotrophs.

Expression of corticotropin releasing factor (CRF) in the enteric nervous system of the jejunum of sheep

&amp;nbsp;Corticotropin releasing factor (CRF), a 41-amino acid neuropeptide widely distributed in the mammalian central nervous system, has been shown to influence several gastrointestinal functions. Recent studies show that CRF released locally from enteric nerves may also underlie alterations in gut function. In this study, immunohistochemisty was applied to demonstrate the presence of CRF in the jejunum of sheep. Using double immunohistochemical staining the co-localization of CRF with vasoactive intestinal peptide (VIP), galanin, tyrosine hydroxylase (TH), neuropeptide&amp;nbsp;Y (NPY) and substance P (SP) was evaluated. The presence of CRF was detected in myenteric neurons (3.6 &amp;plusmn; 0.9%) as well as in submucous neurons (10.5 &amp;plusmn; 1.2%). In the ovine jejunum different numbers of CRF-expressing nerve fibres were detected in myenteric ganglia, submucous ganglia, circular smooth muscle layer, lamina muscularis mucosae and between mucosal glands. None of the CRF-positive enteric neurons and CRF-positive nerve fibres exhibited the presence of TH. CRF-immunoreactive (IR) myenteric neurons widely co-expressed VIP and/or NPY. A minor population of CRF-IR myenteric neurons additionally co-stored SP. Galanin was not present in CRF-IR myenteric neurons. The presence of VIP was observed in the vast majority of CRF-positive submucous neurons. Moderate numbers of CRF-IR sumbucous neurons co-expressing galanin or NPY were also found. The presence of SP in CRF-positive submucous neurons was noted only incidentally. In the circular smooth muscle layer CRF-IR/VIP-IR, CRF-IR/NPY-IR as well as CRF-IR/SP-IR nerve fibres were present. In the mucosal layer of the ovine jejunum CRF-IR nerve fibres co-stored additionally VIP, galanin, NPY or SP. This present study provides for the first time evidence that CRF present in different subclasses of enteric neurons may influence certain activities of the ovine jejunum. Co-localization studies indicate that VIP, galanin, SP and NPY functionally co-operate with CRF in the jejunum of the sheep. &amp;nbsp;

Loss of sensory and noradrenergic innervation in benign colorectal adenomatous polyps – a putative role of semaphorins 3F and 3A

Nerve fibers can exert trophic/anti-trophic effects on epithelial cells. Substance P (SP) is a pro-proliferative neuropeptide, whereas sympathetic noradrenaline is anti-proliferative at high concentrations. Density of noradrenergic and sensory nerve fibers and presence of nerve repellent factors specific for noradrenergic (semaphorin 3F) and sensory nerve fibers (semaphorin 3A) were investigated in colorectal adenomas. The pedunculus was innervated by noradrenergic fibers, whereas the mucosa was sparsely innervated. The control submucosa compared with control mucosa demonstrated increased density of noradrenergic fibers. Control tissue was much better innervated than the polyp. This was accompanied by strong expression of semaphorin 3F in epithelial cells. Density of sensory SP+ nerve fibers was higher in control colon mucosa compared with polyp mucosa, and SP+ cell clusters and semaphorin 3A-positive cells appeared in the intercrypt space in polyps, but not in control tissue. This study demonstrated a marked loss of noradrenergic and sensory nerve fibers in polyp mucosa, which was associated with a strong increase of semaphorin 3F and 3A. Up-regulation of the sympathetic repellent semaphorin 3F in the polyps possibly triggers sympathetic repulsion and polyp growth due to the loss of anti-proliferative noradrenaline and presence of SP from local SP+ cells.

Pituitary Adenylate Cyclase‐Activating Peptide (PACAP) and Substance P (SP) induce the release of Brain‐Derived Neurotropic Factor (BDNF) from the longitudinal muscle layer of the intestine

BDNF has been implicated in neuronal development and plasticity. While BDNF is expressed in neural and non-neuronal cells, the expression and secretion of BDNF in gastrointestinal smooth muscle is not well defined. Aim To determine if BDNF is expressed and secreted from intestinal smooth muscle. Methods BDNF expression in intestinal segments was evaluated immunohistochemically. Longitudinal muscle cells were cultured for 24 to 48 hours in the absence or presence of SP or PACAP (10 to 100 nM); BDNF content in cells and secretion into media was measured by western blot and ELISA. Results Immunohistochemically, BDNF was identified primarily in the longitudinal rather than circular muscle layer of mouse and rabbit intestine. Western blot analysis of BDNF from longitudinal muscle cultures confirmed basal BDNF expression and demonstrated time- and concentration-dependent increase in BDNF content in response to both neuropeptides, with PACAP being more effective than SP. Similar results were obtained for release of BDNF into culture medium. Conclusion In the basal state, intestinal smooth muscle cells from the longitudinal layer express and secrete BDNF. Contractile and relaxant neuropeptides modulate this expression and secretion suggesting crosstalk between enteric neurons and smooth muscle via interplay between neuropeptide transmitters and neurotrophins. Supported by NIDDKD.

Substance P primes lipoteichoic acid‐ and Pam3CysSerLys4‐mediated activation of human mast cells by up‐regulating Toll‐like receptor 2

Substance P (SP) is a neuropeptide with neuroimmunoregulatory activity that may play a role in susceptibility to infection. Human mast cells, which are important in innate immune responses, were analysed for their responses to pathogen-associated molecules via Toll-like receptors (TLRs) in the presence of SP. Human cultured mast cells (LAD2) were activated by SP and TLR ligands including lipopolysaccharide (LPS), Pam3CysSerLys4 (Pam3CSK4) and lipoteichoic acid (LTA), and mast cell leukotriene and chemokine production was assessed by enzyme-linked immunosorbent assay (ELISA) and gene expression by quantitative PCR (qPCR). Mast cell degranulation was determined using a β-hexosaminidase (β-hex) assay. SP treatment of LAD2 up-regulated mRNA for TLR2, TLR4, TLR8 and TLR9 while anti-immunoglobulin E (IgE) stimulation up-regulated expression of TLR4 only. Flow cytometry and western blot confirmed up-regulation of TLR2 and TLR8. Pretreatment of LAD2 with SP followed by stimulation with Pam3CSK4 or LTA increased production of leukotriene C4 (LTC(4) ) and interleukin (IL)-8 compared with treatment with Pam3CSK4 or LTA alone (>2-fold; P<0·01). SP alone activated 5-lipoxygenase (5-LO) nuclear translocation but also augmented Pam3CSK4 and LTA-mediated 5-LO translocation. Pam3CSK4, LPS and LTA did not induce LAD2 degranulation. SP primed LTA and Pam3CSK4-mediated activation of JNK, p38 and extracellular-signal-regulated kinase (ERK) and activated the nuclear translocation of c-Jun, nuclear factor (NF)-κB, activating transcription factor 2 (ATF-2) and cyclic-AMP-responsive element binding protein (CREB) transcription factors. Pretreatment with SP followed by LTA stimulation synergistically induced production of chemokine (C-X-C motif) ligand 8 (CXCL8)/IL-8, chemokine (C-C motif) ligand 2 (CCL2)/monocyte chemotactic protein 1 (MCP-1), tumour necrosis factor (TNF) and IL-6 protein. SP primes TLR2-mediated activation of human mast cells by up-regulating TLR expression and potentiating signalling pathways associated with TLR. These results suggest that neuronal responses may influence innate host defence responses.

Age-related changes in substance P-immunoreactive nerve structures of the rat recto-anal region

Abstract The recto-anal region is innervated by extrinsic and intrinsic nerves and a number of neuropeptides including substance P (SP) have been suggested to participate in the regulation of intestinal movements. We examined the age-related changes in the distribution of SP-immunoreactive nerve structures in the distal part of the rat large intestine. Using immunohistochemistry, the presence of SP was studied in fresh tissues from Wistar rats at different ages taken at three sampling sites, the distal rectum, anal canal and internal anal sphincter. In the 15-day old rats the myenteric plexus of the distal rectum and anal canal was well outlined by numerous SP-immunoreactive varicose nerve fibres encircling immunonegative perikarya. In the circular muscle layer, nerve fibres and small nerve bundles ran parallel to the muscle cells, while in the longitudinal muscle layer, only occasional nerve fibres were seen. At the level of the internal anal sphincter, no myenteric ganglia were present. Here, thin varicose fibers ran parallel to the smooth muscle cells. In the 3-month old rats, a larger number of intensely staining SP-immunoreactive nerve fibres were found and in the circular muscle layer, thicker nerve strands were observed. In the 26-month old rats, the density and staining intensity of SP-immunopositive nerve fibres in the myenteric plexus was lower than in the 3-month-old rats. Similar changes in the SP-immunostained fibres in the internal anal sphincter were observed. Degenerative alterations in SP-containing fibres during aging appear to play a role in ano-rectal motility and sphincter control.

Substance P-induced inhibition of Leydig cell steroidogenesis in primary culture

The effect of substance P (SP) on hamster Leydig cell steroidogenesis in primary culture was investigated. Purified Leydig cells were cultured with or without SP for 24 h. The levels of testosterone and progesterone in the culture media were 174 +/- 20 pg ml-1 and 105 +/- 12 pg ml-1, respectively. In the presence of SP (10(-7) mol l-1), testosterone concentration significantly decreased to 123 +/- 19 pg ml-1, e.g. with 29.3%. By contrast, at the concentration used, SP had no effect on progesterone secretion. The possible molecular mechanism of the SP action on Leydig cell function was discussed. The reported results indicate that SP can modulate Leydig cell steroidogenesis in culture.

Substance P potentiates TGFbeta-1 production in lung epithelial cell lines.

Transforming growth factor-beta (TGF-beta) is one of the most important cytokines implicated in growth, differentiation, repair and also the pathogenesis of the lung fibrosis by its stimulatory effect on extracellular matrix deposition. Pulmonary epithelial cells are considered as a source of TGF-beta in lung. Substance P (SP), as a neuroimmunomodulator has elevated levels in inflamed airways and although it has significant role in the pathogenesis of the lung fibrosis, but its effect on transforming growth factor -beta (TGF-beta) production of the lung epithelial cells (and so its regulatory potential) remains unclear. In this study TGFbeta-1 levels in supernatants of the normal (BEAS-2B) and cancerous (A549) lung epithelial cell line cultures at the presence of various concentrations of SP were examined and MTT assay was performed to evaluate cells viability. We have observed that SP (without any other stimulator) significantly augments TGF-beta production of both BEAS and A54 cells and this effect is inhibited by NK1-receptor antagonist (CP-96345). We have also observed that the viability of cells did not significantly affect at the presence of SP. It can be concluded that SP can directly modulate the release of TGF-beta from human bronchial epithelial cell line and thereby participates in various lung functions or pathologic conditions.

An immunohistochemical study on a unique colocalization relationship between substance P and GABA in the central nucleus of amygdala

Substance P (SP) is a neuropeptide contained in axon terminals. Various classical neurotransmitters coexist with SP in mammalian brains, but there has been no information on the colocalizing substances in the central nucleus of amygdala (CeA), where both SP and its specific receptor are highly concentrated. The present study aimed at determining the colocalizing neurotransmitter in SP terminals in CeA by multi-label immunohistochemistry combined with digitized quantitative analysis. Unexpectedly, most of SP-containing boutons did not show immunoreactivities for any of the transmitters or their marker proteins examined (GABA, glycine, glutamate, acetylcholine, serotonin, or dopamine). Electron microscopy demonstrated small clear vesicles in addition to dense core vesicles within SP-positive terminals that formed symmetrical synapses, indicating the presence of some classical neurotransmitter, most likely GABA. Therefore tissues were fixed by zinc–aldehyde to enhance immunoreactivity for a low level of glutamic acid decarboxylase (GAD), the GABA synthetic enzyme. This led to weak but consistent labeling for GAD in the majority of SP-positive boutons in CeA. By contrast, definite GAD-immunoreactivity was confirmed in SP-containing boutons in the substantia nigra pars reticulata even in specimens treated with a conventional fixative, indicating that negligible GAD labeling in CeA is not ascribed to methodological problems such as interference by the presence of SP but actually reflects low GAD content. These data suggest a unique mode of synaptic transmission at amygdalar SP-containing terminals where slowly-acting SP is concentrated but both GABA and its synthetic enzyme are maintained at low levels, possibly underlying long-lasting responses in emotions.

The effect of substance P on nitric oxide production by HSV-1 infected macrophages

The regulatory role of neuro-immune pathways in various infectious disease states is an interesting area of investigation due to its important therapeutic potential. In this study the effect of substance P (SP) on nitric oxide (NO) production of HSV-infected macrophages and its relation with incubation time and also the cytopathic effect of virus in the presence of SP are considered. As we have observed, substance P enhances NO production both in HSV- and mock-infected macrophages and this effect has been diminished or disappeared after 24 h. It has also been observed that SP enhances the cytopathic effect of HSV in a time dependent manner with the most effect at 10 h after stimulation with SP. We have also observed that HSV-1 cytopathic effect has not been significantly affected by pretreatment of macrophages with SP (2, 10 or 24 h before HSV infection). It can be concluded that the neurotransmitter SP has a significant role in modulating macrophage function and thus innate immunity processes in healthy and HSV-infected cells.

Substance P stimulates release of RANKL via COX-2 expression in human dental pulp cells

Our previous study found that substance P (SP), a sensory neuropeptide, was expressed in the dental pulp of rats during experimental tooth movement. We examined the effects of SP on the production of prostaglandin (PG) E2 and the receptor activator of nuclear factor- B ligand (RANKL) by human dental pulp fi broblast-like (HDPF) cells. SP was added to cultured HDPF cells at concentrations ranging from of 10(-4) to 10(-12) mol/L. PGE2 and soluble RANKL (sRANKL) levels were determined using enzyme-linked immunosorbent assay kits. Gene expression was confi rmed by RT-PCR analysis. Pit formation assays using dentin slices were carried out to examine the effect of SP on osteoclastogenesis. The levels of PGE2 and sRANKL increased in the presence of SP, though the increases were greater in the experimental groups in both a time- and concentration-dependent manner, and the increase of RANKL was partially mediated by PGE2. The gene expression of cyclooxygenase (COX)-2 and RANKL was up-regulated, and conditioned medium samples obtained from HDPF cells treated with SP induced bone resorption. SP stimulated the production of PGE2 and RANKL, and promoted bone resorption. Therefore, SP may be involved in pulpal inflammation and root resorption during orthodontic tooth movement.