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The distribution and chemical coding of neurons supplying urinary bladder in the male pig were studied in the sympathetic chain ganglia, inferior mesenteric ganglia and anterior pelvic ganglia. The combined retrograde tracing and immunohistochemistry for tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), neuropeptide Y (NPY), somatostatin(SOM), galanin (GAL), vasoactive intestinal polypeptide(VIP), nitric oxide synthase (NOS), calcitonin gene related peptide (CGRP), substance P (SP), choline acetyltransferase (ChAT) and vesicular acetylcholine transporter(VAChT) were applied in the experiment. Bladder projecting neurons were found in all the ganglia studied. The majority of sympathetic ganglia neurons (inferior mesenteric ganglia and sympathetic chain ganglia) expressed immunoreactivity(IR) to DBH. In sympathetic chain ganglia these neurons simultaneously expressed NPY, GAL or VAChT,while in inferior mesenteric ganglia they contained NPY, SOM and/or GAL. A small number of these bladder projecting neurons was VAChT-IR and some contained NPY. In the pelvic ganglia bladder-projecting neurons formed two populations: DBH- and VAChT-IR. Some of DBH-IR neurons contained IR to NPY, SOM or GAL, while VAChTIR neurons were NPY-, SOM- or NOS-IR. The results indicate that sympathetic ganglia contain mainly adrenergic neurons,while pelvic ganglia contain both adrenergic and cholinergic neurons. All these neurons contain typical combinations of neuropeptides.

The pig, as an omnivorous animal, seems to be especially valuable species in "gastrointestinal" experiments. The importance of the pylorus in the proper functioning of the digestive tract is widely accepted. Although it is commonly known that sensory innervation plays an important role in the regulation of gastric activity and gastrointestinal tissue resistance, there is complete lack of data on the extrinsic afferents projecting to the swine pylorus. The present experiment has been designed to discover the precise localization and neurochemical properties of the primary sensory neurons projecting to the porcine pylorus. Combined retrograde tracing technique and double immunocytochemistry were applied in five piglets. An additional RT-PCR reaction was used to confirm the presence of all investigated neurotransmitters in the studied ganglia. Traced neurons were localized within the bilateral nodose ganglia of the vagus and bilateral dorsal root ganglia spreading from Th4 to L1. Fast Blue-positive afferents expressed immunoreactivity to substance P, calcitonin gene-related peptide, neuronal isoform of nitric oxide synthase, and galanin. In the vagal and spinal ganglia, the percentages of traced neurons immunoreactive to these substances were 54.8, 10.7, 49.6, 7.4 % and 22.2, 75.5, 95.2 %, respectively, and the solitary perikarya were Gal immunoreactive. The presence of all substances studied in the ganglion tissue was confirmed by RT-PCR technique.

The aim of our study was to localize and define immunocytochemical characteristic of the dorsal motor nucleus of the vagus (DMX) neurons projecting to the porcine stomach prepyloric region in the physiological state and after gastric partial resection. To identify the stomach-projecting perikarya, the neuronal retrograde tracer--Fast Blue (FB) was injected into the studied region of control and resection group (RES). In the RES group, on 22nd day after FB injection, the partial resection of the stomach region previously injected with FB was performed. Sections were immunostained with ChAT, pituitary adenylate cyclase-activating peptide (PACAP), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), galanin (GAL), substance P (SP), leu-enkephalin (LENK), and cocaine- and amphetamine-regulated transcript (CART). In the DMX of control and RES group, the stomach-projecting perikarya were found in the entire extent of the nucleus bilaterally. Within control animals, 30.08 ± 1.97 % of the gastric DMX perikarya expressed PACAP, while other substances were found only in the neuronal fibers. In the RES group DMX, PACAP was found in 45.58 ± 2.2 %, VIP in 28.83 ± 3.63 %, NOS in 21.22 ± 3.32 %, and GAL in 5.67 ± 1.49 % of the FB-labeled gastric perikarya. Our data implicate PACAP, VIP, NOS, and GAL as neuronal survival promoting substances and the CART-, LENK-, SP- NOS-, and GAL-immunoreactive processes in control of the gastric vagal neurons in the pig.

The study determined the distribution and immunohistochemical coding of the sensory neurons innervating the male pig urinary bladder. Retrograde tracer Fast Blue was injected bilaterally into the bladder trigone, base or dome. The presence of neuropeptide Y (NPY), somatostatin (SOM), galanin (GAL), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), calcitonin gene-related peptide (CGRP) and substance P (SP) were studied with immunofluorescence. Fast Blue-positive neurons were localized bilaterally in dorsal root ganglia from L1 to L6 and from S3 to S4 with specific differences regarding the injection site. The number of Fast Blue-positive neurons was higher in the right ganglia. Immunohistochemistry revealed that sensory neurons innervating the urinary bladder trigone, base and dome displayed immunoreactivities to CGRP, SP, NOS, GAL and SOM. Differences in the neuropeptide content were observed between the Fast Blue-positive neurons in lumbar and sacral ganglia. Taken together, these data indicate that the lumbar and sacral pathways probably play different roles in sensory transmission from the urinary bladder trigone, base and dome.

The main goal of our research was to study the possible alterations of the chemical coding of the dorsal motor vagal nucleus (DMX) neurons projecting to the porcine stomach prepyloric region following prolonged acetylsalicylic acid supplementation. Fast Blue (FB) was injected into the studied area of the stomach. Since the seventh day following the FB injection, acetylsalicylic acid (ASA) was given orally to the experimental gilts. All animals were euthanized on the 28th day after FB injection. Medulla oblongata sections were then processed for double-labeling immunofluorescence for choline acetyltransferase (ChAT), pituitary adenylate cyclase-activating peptide (PACAP), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), galanin (GAL), substance P (SP), leu enkephalin (LENK), and cocaine- and amphetamine-regulated transcript (CART). In the control DMX, only PACAP was observed in 30.08 ± 1.97 % of the FB-positive neurons, while VIP, NOS, GAL, SP, LENK, and CART were found exclusively in neuronal processes running between FB-labeled perikarya. In the ASA DMX, PACAP was revealed in 49.53 ± 5.73 % of traced vagal perikarya. Moreover, we found de novo expression of VIP in 40.32 ± 7.84 %, NOS in 25.02 ± 6.08 %, and GAL in 3.37 ± 0.85 % of the FB-labeled neurons. Our results suggest that neuronal PACAP, VIP, NOS, and GAL are mediators of neural response to aspirin-induced stomach inflammatory state.

Background: The substance P pathway modulates neuroimmune interactions during intestinal inflammation., Aims: To analyse mucosal expression and genetic variants of the genes coding for substance P, neurokinin-1 receptor and neutral endopeptidase in patients with inflammatory bowel disease., Methods: qRT-PCR was used to analyse mRNA levels in matched, paired samples of inflamed colonic mucosa and adjacent non-inflamed endoscopic tissue from 26 Crohn's disease and 25 ulcerative colitis patients. Allele and genotype frequencies of tag-SNPs were determined in 908 Crohn's disease, 929 ulcerative colitis, and 853 controls. Expression levels and genotype distributions were examined within patients' clinical sub-phenotypes., Results: All 3 evaluated genes were overexpressed in inflamed tissues from Crohn's disease (P=0.033, P=4×10(-5), P=0.001), while in ulcerative colitis only higher levels of the gene coding for neutral endopeptidase were statistically significant (P=2.5×10(-5)). Smoking habit and perianal disease were significantly associated with substance P (P=0.002) and neurokinin-1 receptor levels (P=0.02) in Crohn's disease. Neutral endopeptidase rs701109 variant was associated with inflammatory bowel disease (Crohn's disease: P=0.022; ulcerative colitis: P=0.045), and with the need for colectomy in ulcerative colitis (P=0.008, OR=2.46, 95% CI=1.27-4.76)., Conclusions: Genetic variants of the gene coding for neutral endopeptidase might affect the neuroimmune interaction during intestinal inflammation and influence clinical sub-phenotypes in patients with inflammatory bowel disease., (Copyright © 2013 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Aim: To evaluate the therapeutic effect of Shugan-decoction (SGD) on visceral hyperalgesia and colon gene expressions using a rat model., Methods: Ninety-six adult male Wistar rats were randomized into six equal groups for assessment of SGD effects on psychological stress-induced changes using the classic water avoidance stress (WAS) test. Untreated model rats were exposed to chronic (1 h/d for 10 d consecutive) WAS conditions; experimental treatment model rats were administered with intragastric SGD at 1 h before WAS on consecutive days 4-10 (low-dose: 0.1 g/mL; mid-dose: 0.2 g/mL; high-dose: 0.4 g/mL); control treatment model rats were similarly administered with the irritable bowel syndrome drug, dicetel (0.0042 g/mL); untreated normal control rats received no drug and were not subjected to the WAS test. At the end of the 10-d WAS testing period, a semi-quantitative measurement of visceral sensitivity was made by assessing the abdominal withdrawal reflex (AWR) to colorectal balloon-induced distension (at 5 mmHg increments) to determine the pain pressure threshold (PPT, evidenced by pain behavior). Subsequently, the animals were sacrificed and colonic tissues collected for assessment of changes in expressions of proteins related to visceral hypersensitivity (transient receptor potential vanilloid 1, TRPV1) and sustained visceral hyperalgesia (substance P, SP) by immunohistochemistry and real-time polymerase chain reaction. Inter-group differences were assessed by paired t test or repeated measures analysis of variance., Results: The WAS test successfully induced visceral hypersensitivity, as evidenced by a significantly reduced AWR pressure in the untreated model group as compared to the untreated normal control group (190.4 ± 3.48 mmHg vs 224.0 ± 4.99 mmHg, P < 0.001). SGD treatments at mid-dose and high-dose and the dicetel treatment significantly increased the WAS-reduced PPT (212.5 ± 2.54, 216.5 ± 3.50 and 217.7 ± 2.83 mmHg respectively, all P < 0.001); however, the low-dose SGD treatment produced no significant effect on the WAS-reduced PPT (198.3 ± 1.78 mmHg, P > 0.05). These trends corresponded to the differential expressions observed for both TRPV1 protein (mid-dose: 1.64 ± 0.08 and high-dose: 1.69 ± 0.12 vs untreated model: 3.65 ± 0.32, P < 0.001) and mRNA (0.44 ± 0.16 and 0.15 ± 0.03 vs 1.39 ± 0.15, P < 0.001) and SP protein (0.99 ± 0.20 and 1.03 ± 0.23 vs 2.03 ± 0.12, P < 0.01) and mRNA (1.64 ± 0.19 and 1.32 ± 0.14 vs 2.60 ± 0.33, P < 0.05). These differential expressions of TRPV1 and SP related to mid- and high-dose SGD treatments were statistically similar to the changes induced by dicetel treatment. No signs of overt damage to the rat system were observed for any of the SGD dosages., Conclusion: Shugan-decoction can reduce chronic stress-induced visceral hypersensitivity in rats, and the regulatory mechanism may involve mediating the expressions of TRPV1 and SP in colon tissues.

This study was designed to investigate the alterations of substance P (SP) and its correlation with apoptosis of the retinal neurons in diabetic rats. The study was carried out with diabetic rats induced by streptozotocin. Changes of SP and its mRNA were examined using enzyme-linked immunosorbent assay and reverse transcription polymerase chain reaction. The effect of restoration of SP level by capsaicin (20mg/kg, s.c.) on the apoptosis of the retinal cells was studied. The apoptosis was evaluated by change of ratio of the apoptotic cells and caspase-3 activity in the retina. It was found that increase in apoptosis of retinal cells, by 3.5 fold of control, was accompanied by reduction of SP, by 28% in protein and 32% in the mRNA in the retina at 10 weeks of induction of diabetes, compared to the controls. Capsaicin significantly elevated endogenous SP, by 29% in the mRNA and 17% in protein in the retina, with marked inhibition of the apoptosis and the activity of caspase-3 in the diabetic rats. Induction of diabetes leads to the increase of cell apoptosis and the decrease of SP in the retina. The reduction of the endogenous SP and the increase of the cell apoptosis in the retina of the diabetic rats were reversed by pretreatment with capsaicin. Restoration of SP in the retina may be a novel option for prevention of the retinal injury during development of diabetes., (© 2013.)

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.)

Study Design: Laboratory study., Objective: The aims of this study were as follows: (1) to confirm that Substance P (SP) is expressed by nucleus pulposus (NP) and annulus fibrosus (AF) cells; (2) to determine the effect of SP on expression of inflammatory mediators in human disc cells and the effect of inflammatory mediators on the expression of SP; and (3) to characterize the relative expression of SP receptor isoforms in disc tissue and describe whether exposure to SP changes receptor expression., Summary of Background Data: SP, classically described as a neurotransmitter, acts as an inflammatory regulator in other tissue types, but its role within the intervertebral disc has not been characterized., Methods: Human AF and NP cells from 7 individuals were expanded in monolayer and maintained in alginate bead culture. Cells were treated with SP or interleukin (IL)-1β/tumor necrosis factor-α (TNF-α). After treatment, the cells were recovered and then RNA was isolated and transcribed into cDNA. Quantitative reverse-transcriptase polymerase chain reaction was performed to evaluate expression of inflammatory mediators and SP and its receptors., Results: Disc cells treated with SP demonstrated significant upregulation of IL-1β, IL-6, and IL-8 in NP and AF cells whereas significant upregulation of RANTES and TNF occurred only in the AF cells. AF and NP cells expressed SP at low levels; expression did not change significantly with SP treatment but was significantly upregulated after treatment with IL-1β/TNF-α. Both SP receptor isoforms were expressed by NP and AF cells., Conclusion: SP upregulates inflammatory mediators in disc cells. SP and its receptors were expressed in both NP and AF cells, and expression did not change after treatment with SP but increased after treatment with IL-1β/TNF-α. SP likely acts in an autocrine or paracrine manner in intervertebral disc cells and may be involved in "crosstalk" between disc cells and neurons, providing a potential mechanism for transmission of painful discogenic stimuli.

Many data suggest the deep involvement of the substance P (SP)/neurokinin (NK)-1 receptor system in cancer: (1) Tumor cells express SP, NK-1 receptors and mRNA for the tachykinin NK-1 receptor; (2) Several isoforms of the NK-1 receptor are expressed in tumor cells; (3) the NK-1 receptor is involved in the viability of tumor cells; (4) NK-1 receptors are overexpressed in tumor cells in comparison with normal ones and malignant tissues express more NK-1 receptors than benign tissues; (5) Tumor cells expressing the most malignant phenotypes show an increased percentage of NK-1 receptor expression; (6) The expression of preprotachykinin A is increased in tumor cells in comparison with the levels found in normal cells; (7) SP induces the proliferation and migration of tumor cells and stimulates angiogenesis by increasing the proliferation of endothelial cells; (8) NK-1 receptor antagonists elicit the inhibition of tumor cell growth; (9) The specific antitumor action of NK-1 receptor antagonists on tumor cells occurs through the NK-1 receptor; (10) Tumor cell death is due to apoptosis; (11) NK-1 receptor antagonists inhibit the migration of tumor cells and neoangiogenesis. The NK-1 receptor is a therapeutic target in cancer and NK-1 receptor antagonists could be considered as broad-spectrum antitumor drugs for the treatment of cancer. It seems that a common mechanism for cancer cell proliferation mediated by SP and the NK-1 receptor is triggered, as well as a common mechanism exerted by NK-1 receptor antagonists on tumor cells, i.e. apoptosis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The present study examines the response of colon-projecting neurons localized in the inferior mesenteric ganglia (IMG) to axotomy in the pig animal model. In all animals (n = 8), a median laparotomy was performed under anesthesia and the retrograde tracer Fast Blue was injected into the descending colon wall. In experimental animals (n = 4), the descending colon was exposed and the bilateral caudal colonic nerves were identified and severed. All animals were euthanized and the inferior mesenteric ganglia were harvested and processed for double-labeling immunofluorescence for calbindin-D28k (CB) in combination with either tyrosine hydroxylase (TH), neuropeptide Y (NPY), somatostatin (SOM), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), Leu-enkephalin (LENK), substance P, vesicular acetylcholine transporter, or galanin. Immunohistochemistry revealed significant changes in the chemical coding pattern of injured inferior mesenteric ganglion neurons. In control animals, Fast Blue-positive neurons were immunoreactive to TH, NPY, SOM, VIP, NOS, LENK, and CB. In the experimental group, the numbers of TH-, NPY-, and SOM-expressing neurons were reduced, whereas the number of neurons immunoreactive to LENK was increased. Our data indicate that the colon-projecting neurons of the porcine IMG react to the axotomy in a similar, but not an identical manner in a comparison to other species, especially rodents. Further studies are needed to elucidate the detailed factors/mechanisms involved in the response to nerve injury.

Objective: To observe the effect of acupuncture of "Zusanli" [ST 36, Ho (Sea)-acupoint] and "Zhongwan" (CV 12, front Mu-acupoint) on the expression of hypothalamic gonadotropin-releasing hormone (GnRH) mRNA and SP mRNA in stress-induced gastric ulcer (GU) rats., Methods: Sixty male Wistar rats were randomly divided into normal control group, simple bundling group, GU model group, CV 12 group, ST 36 group and CV 12 + ST 36 (Ho- and front Mu-acupoint combination) group, with 10 cases in each group. GU model was established by binding the rat's four limbs to an animal board and to immerse it to cool water for 24 h. Manual acupuncture stimulation was applied to CV 12, bilateral ST 36 or CV 12 + ST 36 for 20 min, once daily for 2 days. The GU index was evaluated by using Guth's method (1979). GnRH mRNA and SP mRNA expression of the hypothalamus tissue was detected by reverse transcription-polymerase chain reaction (RT-PCR)., Results: Compared with the normal group, the GU index values in the simple bundling group and the model group were significantly higher (P < 0.01); while in comparison with the model group, the GU index values were significantly decreased in the CV 12, ST 36 and CV 12 + ST 36 groups (P < 0.05, P < 0.01). Following stress stimulation, hypothalamic GnRH mRNA expression levels in the simple bundling group and the model group were remarkably increased (P < 0.05, P < 0.01), whereas those of hypothalamic SP mRNA expression had no apparent changes in these two groups (P > 0.05). After manual acupuncture intervention, hypothalamic GnRH mRNA expression levels in the CV 12, ST 36 and CV 12 + ST 36 groups were evidently down-regulated and SP mRNA expression levels in the ST 36 and CV 12+ ST 36 groups were remarkably upregulated in comparison with the model group (P < 0.01). No significant differences were found between the CV 12 and ST 36 groups in the GU index, among the CV 12, ST 36 and CV 12 + ST 36 groups in GnRH mRNA expression, and between the CV 12 and model groups in the expression levels of SP mRNA (P > 0.05)., Conclusion: Acupuncture of CV 12 and ST 36 can relieve stress-induced gastric ulcer in the rat, which is closely with its effect in down-regulating hypothalamic GnRH mRNA expression.

Zebrafish has emerged as an important vertebrate animal model for the study of human diseases and for developmental studies in mammals. Since there are few studies of the tachykinin 1 gene (TAC1), precursor of substance P (SP), in relation to embryonic development, we aimed to study the expression of SP transcript (mRNA) and determine the influence of cocaine and opioid receptors on the expression of this neuropeptide. In order to analyse the spatial and temporal SP mRNA expression in zebrafish, we cloned - based on human TAC1 sequence - the sequence that originates SP. Phylogenetic analyses of the precursor of SP, revealed an alignment in the fish cluster, with a clear distinction from other species (amphibians, birds and mammals). Real time PCR (qPCR) results showed that SP mRNA was expressed in several stages of embryonic development, where it increased progressively from gastrula-8hpf (hour post-fertilisation) to the end of the embryogenesis-72hpf. SP mRNA was expressed mainly in the spinal cord in embryos at 20-30hpf, whereas at 36, 42 and 48hpf embryos SP mRNA was expressed mainly in the CNS telencephalon, diencephalon, hypothalamus, rhombomeres, epiphysis and in peripheral areas (heart and somites). Exposure of embryos to 1.5μM cocaine altered the SP mRNA expression at 24 (increasing) and 48hpf (decreasing). We also report that knockdown of μ-opioid receptor induced an increase of SP mRNA expression while the knockdown of the two delta opioid receptors did not produce changes in SP mRNA expression. In conclusion, SP mRNA in zebrafish is expressed during embryonic development in the CNS and peripherally, suggesting that SP would play a critical role during embryogenesis. Furthermore, cocaine exposure and the knockdown of μ-opioid receptor affect the SP mRNA expression. These observations can be important in the pain and addiction field where SP is involved., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Most investigations related to the characterisation of the enteric nervous system (ENS) are pivoted on the intestine of small rodents, but few studies are available on the ENS of wild or 'unconventional' rodents. Anti-PGP 9.5 and anti-Hu antibodies were utilised to recognise the distribution pattern of neuronal cell bodies and fibres of the ileum of the Persian squirrel (Sciurus anomalus) ENS. The percentages of subclasses of enteric neurones in the total neuronal population were investigated by neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), calcitonin gene-related peptide (CGRP), substance P (SP), and calbindin (CALB). Myenteric plexus (MP) and submucosal plexus (SMP) neurones showing nNOS immunoreactivity (IR) were 41±4% and 11±6%, respectively, whereas cells expressing ChAT-IR were 56±9% and 74±16%, respectively. nNOS-IR was co-expressed by 21±2% and 9±4% of the MP and SMP cholinergic neurones, respectively, whereas the nNOS-IR MP and SMP neurones co-expressing ChAT-IR were 86±6% and 89±2%, respectively. CGRP-IR and SP-IR were expressed, respectively, by 13±5% and 6±3% of MP and 18±2% and 2±2% of SMP neurones. CALB-IR was expressed by 22±8% and 56±14% of MP and SMP neurones, respectively. MP and SMP cholinergic neurones co-expressed nNOS-IR (21±2% and 9±4%, respectively) and a very high percentage of nNOS-IR neurones showed ChAT-IR (86±6% and 89±2%, respectively). MP and SMP CALB-IR neurones co-expressed ChAT-IR (100% and 63±11%, respectively) and CGRP-IR (89±5% and 26±7%, respectively). Our data might contribute to the neuroanatomical knowledge of the gastrointestinal tract in exotic mammals and provide a comparison with the available data on other mammals., (© 2012 Blackwell Verlag GmbH.)

Background: Approximately 90% of patients who die of Prostate Cancer (PCa) have bone metastases, which promote a spectrum of osteoblastic, osteolytic or mixed bone responses. Numerous secreted proteins have been reported to promote osteoblastic or osteolytic bone responses. We determined whether previously identified and/or novel proteins were associated with the osteoblastic or osteolytic response in clinical specimens of PCa bone metastases., Methods: Gene expression was analyzed on 14 PCa metastases from 11 patients by microarray profiling and qRT-PCR, and protein expression was analyzed on 33 PCa metastases from 30 patients by immunohistochemistry on highly osteoblastic and highly osteolytic bone specimens., Results: Transcript and protein levels of BMP-2, BMP-7, DKK-1, ET-1, and Sclerostin were not significantly different between osteoblastic and osteolytic metastases. However, levels of OPG, PGK1, and Substance P proteins were increased in osteoblastic samples. In addition, Emu1, MMP-12, and sFRP-1 were proteins identified with a novel role of being associated with either the osteoblastic or osteolytic bone response., Conclusions: This is the first detailed analysis of bone remodeling proteins in human specimens of PCa bone metastases. Three proteins not previously shown to be involved may have a role in the PCa bone response. Furthermore, our data suggests that the relative expression of numerous, rather than a single, bone remodeling proteins determine the bone response in PCa bone metastases., (Copyright © 2013 Wiley Periodicals, Inc.)

Objective: Substance P, encoded by the Tac1 gene, is involved in neurogenic inflammation and hyperalgesia via neurokinin 1 (NK1) receptor activation. Its non-neuronal counterpart, hemokinin-1, which is derived from the Tac4 gene, is also a potent NK1 agonist. Although hemokinin-1 has been described as a tachykinin of distinct origin and function compared to SP, its role in inflammatory and pain processes has not yet been elucidated in such detail. In this study, we analysed the involvement of tachykinins derived from the Tac1 and Tac4 genes, as well as the NK1 receptor in chronic arthritis of the mouse., Methods: Complete Freund's Adjuvant was injected intraplantarly and into the tail of Tac1(-/-), Tac4(-/-), Tacr1(-/-) (NK1 receptor deficient) and Tac1(-/-/)Tac4(-/-) mice. Paw volume was measured by plethysmometry and mechanosensitivity using dynamic plantar aesthesiometry over a time period of 21 days. Semiquantitative histopathological scoring and ELISA measurement of IL-1β concentrations of the tibiotarsal joints were performed., Results: Mechanical hyperalgesia was significantly reduced from day 11 in Tac4(-/-) and Tacr1(-/-) animals, while paw swelling was not altered in any strain. Inflammatory histopathological alterations (synovial swelling, leukocyte infiltration, cartilage destruction, bone damage) and IL-1β concentration in the joint homogenates were significantly smaller in Tac4(-/-) and Tac1(-/-/)Tac4(-/-) mice., Conclusions: Hemokinin-1, but not substance P increases inflammation and hyperalgesia in the late phase of adjuvant-induced arthritis. While NK1 receptors mediate its antihyperalgesic actions, the involvement of another receptor in histopathological changes and IL-1β production is suggested.

Pancreatic cancer significantly affects the quality of life due to the severe abdominal pain. However, the underlying mechanism is not clear. This study aimed to determine the relationship between Substance P (SP) and pancreatic cancer perineural invasion (PNI) as well as the mechanism of SP mediating pancreatic cancer PNI, which causes pain in patients with pancreatic cancer. Human pancreatic cancer cells and newborn dorsal root ganglions (DRG) were used to determine the expression of SP or NK-1R in pancreatic cancer cells and DRGs cells by QT-PCR and Western blotting. The effects of SP on pancreatic cancer cell proliferation and invasion were analyzed using MTT assay and Transwell Matrigel invasion assay, respectively. Alterations in the neurotropism of pancreatic cancer cells were assessed by coculture system, which mimics the interaction of tumor/neuron in vivo. SP is not only widely distributed in the neurite outgrowth from newborn DRGs but also expressed in MIA PaCa-2 and BxPC-3 cells. NK-1R is found to be overexpressed in the pancreatic cancer cell lines examined. SP induces cancer cell proliferation and invasion as well as the expression of matrix metalloproteinase (MMP)-2 in pancreatic cancer cells, and NK-1R antagonists inhibit these effects. Furthermore, SP promotes neurite outgrowth and the migration of pancreatic cancer cell cluster to the DRGs, which is blocked by NK-1R antagonists in the coculture model. Our results suggest that SP plays an important role in the development of pancreatic cancer metastasis and PNI, and blocking the SP/NK-1R signaling system is a novel strategy for the treatment of pancreatic cancer.

Mechanical stress plays a vital role in maintaining bone architecture. The process by which osteogenic cells convert the mechanical signal into a biochemical response governing bone modeling is not clear, however. In this study, we investigated how Atlantic salmon (Salmo salar) vertebra responds to exercise-induced mechanical loading. Bone formation in the vertebrae was favored through increased expression of genes involved in osteoid production. Fourier transform infrared spectroscopy (FT-IR) showed that bone matrix secreted both before and during sustained swimming had different properties after increased load compared to control, suggesting that both new and old bones are affected. Concomitantly, both osteoblasts and osteocytes in exercised salmon showed increased expression of the receptor nk-1 and its ligand substance P (SP), both known to be involved in osteogenesis. Moreover, in situ hybridization disclosed SP mRNA in osteoblasts and osteocytes, supporting an autocrine function. The functional role of SP was investigated in vitro using osteoblasts depleted for SP. The cells showed severely reduced transcription of genes involved in mineralization, demonstrating a regulatory role for SP in salmon osteoblasts. Investigation of α-tubulin stained osteocytes revealed cilia-like structures. Together with SP, cilia may link mechanical responses to osteogenic processes in the absence of a canaliculi network. Our results imply that salmon vertebral bone responds to mechanical load through a highly interconnected and complex signal and detection system, with SP as a key factor for initializing mechanically-induced bone formation in bone lacking the canaliculi system., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Scant information is available regarding the effects of cisplatin on the expression profile of tachykinin NK(1) receptors and downstream signaling during cisplatin-induced emesis. Cisplatin causes peak early- and delayed-phase emesis in the least shrew at 1-2 and 33 h post-injection. To investigate the expression profile of NK(1) receptor during both emetic phases, we cloned the cDNA corresponding to a ~700 base pairs of mRNA flanked by two stretches of nucleotides conserved among different species and demonstrated that the shrew NK(1) receptor nucleotide sequence shares ~90% sequence identity with the human NK(1) receptor. Of the 12 time-points tested, significant increases in expression levels of NK(1) receptor mRNA in the shrew brainstem occurred at 2 and 28 h post-cisplatin injection, whereas intestinal NK(1) receptor mRNA was increased at 28 h. Shrew brainstem and intestinal substance P mRNA levels also tended to increase during the two phases. Furthermore, expression levels of NK(1) receptor protein were significantly increased in the brainstem at 2, 8, and 33 h post-cisplatin. No change in brainstem 5-HT(3) receptor protein expression was observed. The temporal enhancements in NK(1) receptor protein expression were mirrored by significant increases in the phosphorylation status of the brainstem ERK1/2 at 2, 8, and 33 h post-cisplatin. Phosphorylation of PKA significantly increased at 33rd and 40th hour. Our results indicate associations between cisplatin's peak immediate- and delayed-phase vomiting frequency with increased: (1) expression levels of NK(1) receptor mRNA and its protein level, and (2) downstream NK(1) receptor-mediated phosphorylation of ERK1/2 and PKA signaling., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Visceral hypersensitivity is an important etiology of non-erosive reflux disease (NERD). Calcitonin gene-related peptide (CGRP) and substance P (SP) are involved in the sensitization of afferent neuronal pathways., Aim: The objectives of this study were to evaluate visceral hypersensitivity in NERD patients, investigate the association between visceral hypersensitivity and mucosal expression of SP and CGRP, and assess their involvement in the pathogenesis of NERD., Methods: Twenty-six NERD patients and 12 healthy volunteers were recruited. Intraesophageal balloon distention was performed, and initial perception threshold (IPT) and threshold of discomfort (ToD) were determined. Immunohistochemical staining was used to measure the optical density (OD) of CGRP and SP-reactive levels in esophageal mucosa, and the numbers of CGRP and SP-reactive neural fibers., Results: IPT and ToD were 9.6 ± 4.8 and 12.3 ± 3.2 ml, respectively, in NERD patients, significantly lower than for controls (13.2 ± 7.5 and 21.6 ± 5.7 ml, P < 0.05 and P < 0.01, respectively). Mean OD values for CGRP and SP staining were significantly higher in NERD than for controls (both P < 0.05) and, in NERD, were negatively correlated with IPT and ToD (all P < 0.01). Numbers of CGRP and SP-reactive neural fibers in esophageal submucosa of NERD patients were significantly increased (both P < 0.05)., Conclusions: Expression of esophageal epithelial CGRP and SP is increased, and correlates negatively with perception thresholds in NERD. These findings may aid understanding of peripheral visceral hypersensitivity and the development of new therapeutic approaches for management of NERD.

Substance P (SP) and its receptor, the neurokinin 1 receptor (NK1R), play important roles in transmitting and regulating somatosensory nociceptive information. However, their roles in visceral nociceptive transmission and regulation remain to be elucidated. In the previous study, moderate SP immunoreactive (SP-ir) terminals and NK1R-ir neurons were observed in the dorsal commissural nucleus (DCN) of the lumbosacral spinal cord. Thus we hypothesized that the SP-NK1R system is involved in visceral pain transmission and control within the DCN. The acute visceral pain behaviors, the colon histological changes and the temporal and spatial changes of NK1R-ir structures and Fos expression in the neurons of the DCN were observed in rats following lower colon instillation with 5% formalin. The formalin instillation induced significant acute colitis as revealed by the histological changes in the colon. NK1R internalization in the DCN was obvious at 8 min. It reached a peak (75.3%) at 30 min, began to decrease at 90 min (58.1%) and finally reached the minimum (19.7%) at 3 h after instillation. Meanwhile, formalin instillation induced a biphasic visceral pain response as well as a strong expression of Fos protein in the nuclei of neurons in the DCN. Finally, intrathecal treatment with the NK1R antagonist L732138 attenuated the NK1R internalization, Fos expression and visceral nociceptive responses. The present results suggest that the visceral nociceptive information arising from inflamed pelvic organs, such as the lower colon, might be mediated by the NK1R-ir neurons in the DCN of the lumbosacral spinal cord.

To clarify a role of substance P (SP) in an endogenous pain control mechanism involving the rat striatum, striatal SP release was measured over time by microdialysis following intraplantar injection of 0.4% formalin. A slow-onset but significant increase of SP and neurokinin 1 receptor (NK1R) internalization in the contralateral striatum were observed following the second phase of formalin-induced nociceptive behaviors. Moreover, 60 min after formalin injection, preprotachykinin-A, the SP mRNA, and the immediate early gene cFOS were upregulated in the contralateral striatum. Continuous infusion of SP into the striatum by reverse microdialysis attenuated formalin-induced second phase, but not the first phase, nociceptive behaviors, and hind paw mechanical allodynia. Moreover, these anti-nociceptive effects of SP were completely inhibited by co-treatment with the NK1R antagonist CP96345. Acute microinjection of SP, however, at a dose that was similar to the total dose of SP continuously infused into the striatum, did not affect formalin-induced nociceptive behaviors. These data indicate that striatal NK1R activation leads to pain suppression rather than facilitation. Furthermore, volume transmission of SP in the striatum appears to be indispensable in the mechanism of pain control. Modulation of striatal NK1Rs could prove to be a useful method of inducing analgesia.

The neurotrophic effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on rat sensory neuronal cell line ND7/23 cells were investigated. PACAP caused a concentration-dependent increase in the number of neurite-bearing cells and the expression of the substance P precursor (PPT) mRNA in 24 h. The effects of PACAP were mimicked by vasoactive intestinal polypeptide with lower potency and dibutyryl-cyclic AMP, and inhibited by inhibitors of protein kinase A, ERK kinase or p38 kinase, KT5720, U0126, or SB203580, respectively. In a PPT promoter luciferase reporter assay, the increase of PPT mRNA was the result of an increase in PPT gene transcriptional activity by PACAP. The increasing effects of PACAP on PPT mRNA were similarly observed in primary cultured rat dorsal root ganglion cells. Thus, PACAP could induce differentiation-like phenomena in sensory neurons in a cAMP-, protein kinase A-, ERK kinase-, and p38 kinase-dependent manner. These results provide evidence of the neurotrophic action of PACAP, which may function to rescue damaged neurons or to switch the neuronal phenotype in injured or inflamed sensory neurons.

We have previously demonstrated nerve growth factor (NGF) regulation of pituitary adenylate cyclase-activating polypeptide (PACAP)/receptors in bladder reflex pathways using a transgenic mouse model of chronic NGF overexpression in the bladder using the urothelial-specific uroplakin II promoter. We have now explored the contribution of target-derived NGF in combination with cyclophosphamide (CYP)-induced cystitis to determine whether additional changes in neuropeptides/receptors are observed in micturition reflex pathways due to the presence of additional inflammatory mediators in the urinary bladder. Quantitative PCR was used to determine PACAP/vasoactive intestinal polypeptide (VIP), substance P, galanin, and receptor transcript expression in the urinary bladder (urothelium, detrusor) in mice with overexpression of NGF in the urothelium (NGF-OE) and wild-type (WT) mice with CYP-induced cystitis (4 h, 48 h, and chronic). With CYP-induced cystitis (4 h), WT and NGF-OE mice exhibited similar changes in galanin transcript expression in the urothelium (30-fold increase) and detrusor (threefold increase). In contrast, PACAP, VIP, and substance P transcripts exhibited differential changes in WT and NGF-OE with CYP-induced cystitis. PAC1, VPAC1, and VPAC2 transcript expression also exhibited differential responses in NGF-OE mice that were tissue (urothelium vs. detrusor) and CYP-induced cystitis duration-dependent. Using conscious cystometry, NGF-OE mice treated with CYP exhibited significant (p ≤ 0.01) increases in voiding frequency above that observed in control NGF-OE mice. In addition, no changes in the electrical properties of the major pelvic ganglia neurons of NGF-OE mice were detected using intracellular recording, suggesting that the urinary bladder phenotype in NGF-OE mice is not influenced by changes in the efferent limb of the micturition reflex. These studies are consistent with target-derived NGF and other inflammatory mediators affecting neurochemical plasticity and the reflex function of micturition pathways.

Background: Patients with peritoneal carcinomatosis often report abdominal pain, which is relatively refractory to morphine. It has been considered that a new animal model is required to investigate the mechanism of abdominal pain for the development of optimal treatments for this type of pain., Methods: To prepare a peritoneal carcinomatosis model, highly peritoneal-seeding gastric cancer cells, 60As6, were implanted into the abdominal cavity. The nociceptive modality for pain-related behavior was assessed in terms of withdrawal behavior in response to mechanical stimuli and hunching behavior. Tissue samples from mouse dorsal root ganglia and spinal cord were subject to immunohistochemistry and real-time reverse transcription polymerase chain reaction., Results: Mice with peritoneal dissemination showed significant hypersensitivity of the abdomen to mechanical stimulation and spontaneous visceral pain-related behavior. There was a significant increase in c-Fos-positive cells in the spinal cord in tumor-bearing mice. Those mice exhibited a remarkable increase in substance P-positive neurons in the dorsal root ganglia (control vs. tumor, 15.4 ± 1.1 vs. 24.2 ± 3.6, P < 0.05, n = 3). A significant decreases in μ-opioid receptor expression mainly in substance P-positive neurons was observed in tumor-bearing mice (69.3 ± 4.9 vs. 38.7 ± 0.9, P < 0.05, n = 3), and a relatively higher dose of morphine was required to significantly reverse the abdominal hypersensitivity., Conclusion: Both the up-regulation of substance P and down-regulation of μ-opioid receptor seen in the dorsal root ganglia may be, at least in part, responsible for the abdominal pain-like state associated with peritoneal carcinomatosis.

Acute pancreatitis (AP) has been associated with an up-regulation of substance P (SP) and neurokinin-1 receptor (NK1R) in the pancreas. Increased SP-NK1R interaction was suggested to be pro-inflammatory during AP. Previously, we showed that caerulein treatment increased SP/NK1R expression in mouse pancreatic acinar cells, but the effect of SP treatment was not evaluated. Pancreatic acinar cells were obtained from pancreas of male swiss mice (25-30 g). We measured mRNA expression of preprotachykinin-A (PPTA) and NK1R following treatment of SP (10(-6) M). SP treatment increased PPTA and NK1R expression in isolated pancreatic acinar cells, which was abolished by pretreatment of a selective NK1R antagonist, CP96,345. SP also time dependently increased protein expression of NK1R. Treatment of cells with a specific NK1R agonist, GR73,632, up-regulated SP protein levels in the cells. Using previously established concentrations, pre-treatment of pancreatic acinar cells with Gö6976 (10 nM), rottlerin (5 μM), PD98059 (30 μM), SP600125 (30 μM) or Bay11-7082 (30 μM) significantly inhibited up-regulation of SP and NK1R. These observations suggested that the PKC-ERK/JNK-NF-κB pathway is necessary for the modulation of expression levels. In comparison, pre-treatment of CP96,345 reversed gene expression in SP-induced cells, but not in caerulein-treated cells. Overall, the findings in this study suggested a possible auto-regulatory mechanism of SP/NK1R expression in mouse pancreatic acinar cells, via activation of NK1R. Elevated SP levels during AP might increase the occurrence of a positive feedback loop that contributes to abnormally high expression of SP and NK1R., (© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Studies in mice with targeted deletions of tachykinin genes suggest that tachykinins and their receptors influence emotional behaviors such as aggression, depression and anxiety. Here, we investigated whether TAC1- and TAC4-encoded peptides (substance P and hemokinin-1, respectively) and the neurokinin-1 receptor (NK-1R) are involved in the modulation of sexual behaviors. Male mice deficient for the NK-1R (TACR1 (-/-)) exhibited decreased exploration of female urine in contrast to C57BL/6 control mice and mice deficient for NK-1R ligands such as TAC1 (-/-), TAC4 (-/-) and the newly generated TAC1 (-/-) /TAC4 (-/-) mice. In comparison to C57BL/6 mice, mounting frequency and duration were decreased in male TACR1 (-/-) mice, while mounting latency was increased. Decreased preference for sexual pheromones was also seen in female TACR1 (-/-) mice. Furthermore, administration of the NK-1R-antagonist L-703,606 decreased investigation of female urine by male C57BL/6 mice, suggesting an involvement of NK-1R in urine sniffing behavior. Our results provide evidence for the NK-1R in facilitating sexual approach behavior, as male TACR1 (-/-) mice exhibited blunted approach behavior toward females following the initial interaction compared with C57BL/6 mice. NK-1R signaling may therefore play an important role in pheromone-induced sexual behavior., (© 2012 The Authors. Genes, Brain and Behavior © 2012 Blackwell Publishing Ltd and International Behavioural and Neural Genetics Society.)

Objective: Substance P (SP) and norepinephrine (NE) containing sensory and sympathetic nerve fibers innervate bone and fracture callus. They are involved in controlling vascularization and matrix differentiation during skeletal growth. Both types of nerve fibers are known to modulate growth and metabolic activity of osteoblasts and osteoclasts. The aim of this study was to understand the roles of SP and NE in chondrocyte metabolism and their impact on chondrocyte proliferation, apoptosis, and cell adhesion., Methods: Primary costal chondrocytes were isolated from newborn mice. Micromass and monolayer cell culture regimens were used to analyze the effects of SP and NE on matrix formation, as determined by quantitative polymerase chain reaction and immunohistochemistry. The effects of SP and NE on proliferation, adhesion, and apoptosis of chondrocytes were determined by enzyme-linked immunosorbent assay, bromodeoxyuridine, TUNEL, and morphometric analyses., Results: SP, neurokinin type 1 (NK-1) receptor, α-adrenergic receptor (α-AR), and β-AR were abundantly expressed in primary costal chondrocytes. Stimulation with SP or NE did not affect extracellular matrix formation with respect to types I, II, and IX collagen and aggrecan in micromass pellets. SP dose-dependently increased the rate of proliferation of chondrocytes via the NK-1 receptor, whereas NE decreased the apoptosis rate of chondrocytes by stimulating β-AR. Both neurotransmitters induced the formation of focal adhesion contacts., Conclusion: Transmitters of sympathetic and sensory nerve fibers modulate the metabolic activity of chondrocytes. Endogenous SP, NK-1 receptor, and adrenoceptor expression in chondrocytes implicates as-yet-unknown, presumably trophic, functions of neurotransmitter for skeletal growth and might be of interest for use in cartilage regenerative medicine., (Copyright © 2012 by the American College of Rheumatology.)

Background: Transient receptor potential vanilloid type-1 (TRPV1)-expressing sensory neurons release neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP), which play a crucial role in the pathomechanism of experimental colitis. We investigated whether innervation density and neuropeptide release were responsible for the proximodistal aggravation of murine dextran-sulfate-sodium-salt (DSS) colitis., Methods: Whole mount TRPV1/CGRP immunostained mouse colon preparations were semiquantitatively analyzed. TRPV1 activation by capsaicin and acidic solution (pH 5.1) induced colonic CGRP/SP release, measured by EIA. Single cell quantitative PCR was employed to measure TRPV1 expression levels in DiI-labeled colonic dorsal root ganglion (DRG) neurons. The proximodistal gradient of DSS colitis severity was investigated in WT, CGRP(-/-), SP(-/-), and resiniferatoxin (RTX)-desensitized mice, employing mouse endoscopy, histology, and body weight measurement., Results: TRPV1/CGRP-positive nerve fiber density was increased in the distal colon wall. CGRP/SP release induced by TRPV1 activation from the distal colon was greater than that from the proximal colon. This gradient further increased in colitis. TRPV1 gene expression increased in colonic DRGs projecting to the distal, compared to that in colonic DRGs projecting to the proximal colon, and was further enhanced during colitis. In contrast to WT and CGRP(-/-) mice, SP(-/-) and RTX-desensitized mice showed amelioration of DSS colitis accompanied by a loss of the proximodistal gradient of inflammation., Conclusions: The spatial correlation among increased colonic innervation density, TRPV1 receptor expression, stimulated SP release, and colitis severity suggested that TRPV1/SP-expressing sensory neurons should be considered as a therapeutic target in human ulcerative colitis.

Background/aims: We aimed to analyze substance P (SP) and neprilysin (NEP), the membrane metallopeptidase that degrades SP, in chronic pancreatitis (CP)., Methods: SP and NEP mRNA levels were analyzed by qRT-PCR in tissue samples from 30 patients with CP and 8 organ donors. In addition, SP serum levels were determined before and after surgery in the same patients, by means of a competitive ELISA assay. Genetic and epigenetic analyses of the NEP gene were also performed., Results: SP mRNA expression levels were higher in CP tissues compared to controls (p = 0.0152), while NEP mRNA showed no significant differences between CP and healthy subjects (p = 0.2102). In CP patients, SP serum levels correlated with those in tissue, and after surgical resection SP serum levels were reduced compared to the preoperative values. Failure of NEP to overexpress in CP tissues was associated with significant miR-128a overexpression (p = 0.02), rather than with mutations in the NEP coding region or the presence of hypermethylation sites in the NEP promoter region., Conclusion: Tissue and serum levels of SP were increased in CP, while NEP levels remained unaltered. In an SP/NEP-mediated pathway, it would appear that NEP fails to provide adequate surveillance of SP levels. Failure of NEP to overexpress could be associated with miRNA regulation., (Copyright © 2012 S. Karger AG, Basel.)

Neuropeptide Y (NPY) is an important neuronal element involved in cardiovascular regulation. Since elevated plasma levels of NPY have been observed in numerous pathological situations, this study aimed to determine whether long-term elevated plasma concentrations of NPY could result in aberrant baroreflex sensitivity. Mini-osmotic pump containing NPY (85 μg per 30 days) was subcutaneously implanted between scapulae in male rats for 4 months. The rats treated with NPY showed the following characters compared with control group: (1) attenuated heart rate responding to the increases in mean arterial blood pressure (MABP) induced by phenylephrine, but enhanced heart rate responding to the decreases in MABP induced by sodium nitroprusside; (2) decreased protein levels of substance P (SP) and GluR2, while increased the expression of γ-aminobutyric acid A receptor (GABA(A)R) in brainstem; (3) abdominal obesity indicated by increased body weight and accumulated fat mass in peritoneal cavity; (4) significant increases in total cholesterol, triglycerides, and low density lipoprotein levels in the periphery. These findings indicate that long-term NPY administration in the periphery leads to abnormal baroreflex sensitivity due, at least in part, to the down-regulated expression of SP/GluR2 and elevated expression of GABA(A)R in both protein and RNA levels, which indicate the alternations in glutamate function and GABA action in the nucleus tractus solitarii in NPY-treated rats. Furthermore, long-term NPY administration results in abdominal obesity and dyslipidemia., (Copyright © 2012 S. Karger AG, Basel.)

Background: Unexplained chronic cough is a common condition without specific causes. A hyperreactivity of the cough reflex has been suggested as a mechanism for inducing chronic cough. We hypothesized that nitrosative stress in the upper airway might play a role in cough hypersensitivity by causing neurochemical abnormalities., Methods: Fifty-one patients with unexplained chronic cough and 27 controls were enrolled. A capsaicin cough provocation test was performed to determine cough sensitivity. Nitrosative stress in the upper airway was assessed by quantifying 3-nitrotyrosine (3-NT) immunostaining in nasal epithelial cells (NECs) and measuring nasal nitric oxide (nNO). The effect of NO on airway epithelium was investigated by measuring the levels of substance P (SP) in nasal lavage fluid and evaluating SP expression in airway epithelial cells., Results: Based on the results of the capsaicin test, patients were divided into two groups: a cough hypersensitivity (CHS) group and a normal cough sensitivity (NCS) group. The levels of 3-NT immunoreactivity in NECs were significantly higher in CHS (49 ± 2.9%) than in NCS (27 ± 3.3%) and controls (12 ± 1.6%), a pattern that was also reflected in the values of nNO (350 ± 43, 215 ± 23, and 138 ± 23 ppb in CHS, NCS, and controls, respectively). SP concentration was also elevated in nasal lavage fluids from CHS (746 ± 28 pg/mL) compared with that from NCS (624 ± 40 pg/mL) and controls (526 ± 41 pg/mL). SP expression in airway epithelial cells was greatly enhanced by exposure to NO donor, which was attenuated by pretreatment with either NO scavenger or NO synthase inhibitor., Conclusion: Increased nitrosative stress in the upper airway may play a role in the pathogenesis of unexplained chronic cough with CHS through enhanced secretion of SP.

Background: Extrinsic sensory neurons play a crucial role in aberrant immune responses in colitis. The activation of peptidergic sensory nerve fibres is accompanied by a release of the neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP). SP levels increase whilst CGRP levels decrease in colon specimens from patients with inflammatory bowel disease; thus suggesting the pro- and anti-inflammatory roles, respectively, of these neuropeptides., Methods: Oxazolone (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one) colitis was induced in wild-type (WT), SP and CGRP knockout ((-/-)) mice. CGRP(-/-) mice were treated with the neurokinin 1-receptor antagonist CP-96345 (CP). The permeability of the mouse colon was evaluated by Evans Blue uptake. Cytokines produced by colonic lamina propria mononuclear cells were measured by ELISA., Results: Colons of WT, CGRP(-/-) and SP(-/-) mice showed similar tissue architecture and permeability. SP(-/-) mice were protected against oxazolone colitis, whereas CGRP(-/-) showed increased susceptibility to colitis compared to WT mice. SP(-/-) and CP-treated CGRP(-/-) mice showed no significant body weight loss during the period of sickness in contrast to untreated CGRP(-/-) and WT mice. Decreased production of IL-4, IL-5, and IL-13 by colonic lamina propria mononuclear cells of the protected SP(-/-) mice confirms the crucial role of these cytokines in oxazolone colitis., Conclusion: We demonstrate that the neuropeptides CGRP and SP exert opposing effects in oxazolone colitis and provide further evidence for a prominent neuroimmune association in the gut., (Copyright © 2011 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Chronic stress, as a risk factor for cardiovascular diseases, has been reported to result in elevated plasma neuropeptide Y (NPY) and be highly associated with abnormal cardiac autonomic function. This study aimed to explore the effect of NPY on the chronic stress-induced abnormal baroreceptor reflex sensitivity (BRS). Seven types of recognized stressors were used to develop chronic stress rat model. Subcutaneously implanting ALZET mini-osmotic pumps containing NPY were used to evaluate the action of NPY on the stressed male rats. We found that chronic stress showed no influence on baseline systolic blood pressure (SBP) and heart rate (HR), whereas NPY (85 μg for 30 days) could elevate baseline SBP and induce bradycardia in rats intervened by various stimuli. NPY pretreatment could preserve chronic stress-induced decreases in left ventricular systolic pressure (LVSP) and the maximum rate of change in left ventricular pressure in the isovolumic contraction period (+dp/dt(max)) but has shown no effect on left ventricular end diastolic pressure (LVEDP) and the maximum rate of change in left ventricular pressure in the isovolumic relaxation period (-dp/dt(max)). Notably, chronic stress led to baroreflex oversensitivity indicated by the elevated ratio of Δheart rate (HR)/ Δmean arterial blood pressure (MABP) in rats followed by vasoconstrictor (phenylephrine, PE) or vasodilator (sodium nitroprusside, SNP) administration, which was almost completely reversed by NPY pretreatment. The expressions of substance P (SP) and gamma aminobutyric acid A receptor (GABA(A)R) in nucleus tractus solitarius were increased in chronic stress rats, which were counteracted by NPY pretreatment. We conclude that chronic stress-induced baroreflex hypersensitivity could be blocked by NPY pretreatment. Furthermore, the altered expressions of neurotransmitters and receptors in the brainstem might contribute to this process., (Copyright © 2012 S. Karger AG, Basel.)

Aims: Substance P and neurokinin A (NKA) are sensory nerve neuropeptides encoded by the TAC1 gene. Substance P is a mast cell secretagogue and mast cells are known to play a role in adverse myocardial remodelling. Therefore, we wondered whether substance P and/or NKA modulates myocardial remodelling via a mast cell-mediated mechanism., Methods and Results: Volume overload was induced by aortocaval fistula in TAC1(-/-) mice and their respective wild types. Left ventricular internal diameter of wild-type (WT) fistulas increased by 31.9%; this was prevented in TAC1(-/-) mice (4.2%). Matrix metalloproteinase (MMP) activity was significantly increased in WT fistula mice and was prevented in TAC1(-/-) mice. Myocardial collagen volume fraction was decreased in WT fistula mice; this collagen degradation was not observed in the TAC1(-/-) group. There were no significant differences between any groups in tumour necrosis factor (TNF)-α or cell death. Cardiac mast cells were isolated from rat hearts and stimulated with substance P or NKA. We found that these cells degranulated only to substance P, via the neurokinin-1 receptor. To determine the effect of substance P on mast cells in vivo, volume overload was created in Sprague-Dawley rats treated with the NK-1 receptor antagonist L732138 (5 mg/kg/day) for a period of 3 days. L732138 prevented: (i) increases in cardiac mast cell density; (ii) increased myocardial TNF-α; and (iii) collagen degradation., Conclusions: Our studies suggest that substance P may be important in mediating adverse myocardial remodelling secondary to volume overload by activating cardiac mast cells, leading to increased TNF-α and MMP activation with subsequent degradation of the extracellular matrix.

In rat hippocampal cultures, the preprotachykinin A (PPTA/Tac1) gene, which encodes the neuropeptide substance P, is regulated by the action of lithium. We used reporter gene and expression constructs to demonstrate that this mechanism of action of lithium is mediated via a previously characterised cis-regulatory Ebox element in the proximal promoter, which binds members of the basic Helix-Loop-Helix family of transcription factors. Consistent with this, in hippocampal cells, both the expression of the endogenous gene and the function of this promoter element are differentially regulated by the basic Helix-Loop-Helix factors, upstream stimulatory factor 1 and 2 (USF1/2). In addition, the genes for USF1 and USF2 are differentially regulated by lithium in these cells. Our data implicate USF1 as a major regulator of the action of lithium on the proximal PPTA promoter.

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.)

Background & Aims: The neuropeptides calcitonin gene-related peptide (CGRP) and substance P, and calcium channels, which control their release from extrinsic sensory neurons, have important roles in experimental colitis. We investigated the mechanisms of colitis in 2 different models, the involvement of the irritant receptor transient receptor potential of the ankyrin type-1 (TRPA1), and the effects of CGRP and substance P., Methods: We used calcium-imaging, patch-clamp, and neuropeptide-release assays to evaluate the effects of 2,4,6-trinitrobenzene-sulfonic-acid (TNBS) and dextran-sulfate-sodium-salt on neurons. Colitis was induced in wild-type, knockout, and desensitized mice., Results: TNBS induced TRPA1-dependent release of colonic substance P and CGRP, influx of Ca2+, and sustained ionic inward currents in colonic sensory neurons and transfected HEK293t cells. Analysis of mutant forms of TRPA1 revealed that TNBS bound covalently to cysteine (and lysine) residues in the cytoplasmic N-terminus. A stable sulfinic acid transformation of the cysteine-SH group, shown by mass spectrometry, might contribute to sustained sensitization of TRPA1. Mice with colitis had increased colonic neuropeptide release, mediated by TRPA1. Endogenous products of inflammatory lipid peroxidation also induced TRPA1-dependent release of colonic neuropeptides; levels of 4-hydroxy-trans-2-nonenal increased in each model of colitis. Colitis induction by TNBS or dextran-sulfate-sodium-salt was inhibited or reduced in TRPA1-/- mice and by 2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopro-pylphenyl)-acetamide, a pharmacologic inhibitor of TRPA1. Substance P had a proinflammatory effect that was dominant over CGRP, based on studies of knockout mice. Ablation of extrinsic sensory neurons prevented or attenuated TNBS-induced release of neuropeptides and both forms of colitis., Conclusions: Neuroimmune interactions control intestinal inflammation. Activation and sensitization of TRPA1 and release of substance P induce and maintain colitis in mice., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Objective and Design: To determine whether RBL-2H3 cells have endogenous substance P (SP) expression under immunoglobulin E (IgE)-activated and inactivated conditions, and to ascertain the function of endogenous SP in the antigen-induced degranulation of RBL-2H3 cells., Materials and Methods: SP mRNA and protein expression in both inactivated and 2,4-dinitrophenol (DNP)-specific IgE-activated RBL-2H3 cells were assessed by real-time PCR and immunofluorescence, respectively. Following activation with DNP-specific IgE, the degranulation of RBL-2H3 cells in response to DNP-bovine serum albumin (BSA), with and without endogenous SP expression, was assessed by monitoring the release of the granular enzyme β-hexosaminidase., Results: Endogenous SP mRNA and peptide expression increased in activated RBL-2H3 cells, compared with inactivated RBL-2H3 cells. The small hairpin RNA (shRNA)-mediated knockdown of endogenous SP reduced the degranulation ability of RBL-2H3 cells., Conclusions: Activated RBL-2H3 cells express endogenous SP which plays a role in antigen-induced degranulation.

Background: Mast cells derive from hematopoietic cell precursors and participate in tissue allergic, immune, and inflammatory processes. They secrete many mediators, including preformed TNF, in response to allergic, neuropeptide, and environmental triggers. However, regulation of mast cell degranulation is not well understood., Objective: We investigated the role of mitochondrial dynamics in degranulation of human cultured mast cells., Methods: Human umbilical cord blood-derived mast cells (hCBMCs) and Laboratory of Allergic Diseases 2 (LAD2) mast cells were examined by confocal and differential interference contrast microscopy during activation by IgE/antigen and substance P (SP). Mast cells in control and atopic dermatitis (AD) skin were evaluated by transmission electron microscopy. LAD2 cells were pretreated with mitochondrial division inhibitor, a dynamin-related protein 1 (Drp1) inhibitor, and small interfering RNA for Drp1, which is necessary for mitochondrial fission and translocation. Calcineurin and Drp1 gene expression was analyzed in stimulated LAD2 cells and AD skin biopsies., Results: Stimulation of hCBMCs with IgE/antigen or LAD2 cells with SP leads to rapid (30 minutes) secretion of preformed TNF. Degranulation is accompanied by mitochondrial translocation from a perinuclear location to exocytosis sites. Extracellular calcium depletion prevents these effects, indicating calcium requirement. The calcium-dependent calcineurin and Drp1 are activated 30 minutes after SP stimulation. Reduction of Drp1 activity by mitochondrial division inhibitor and decrease of Drp1 expression using small interfering RNA inhibit mitochondrial translocation, degranulation, and TNF secretion. Mitochondrial translocation is also evident by transmission electron microscopy in skin mast cells from AD biopsies, in which gene expression of calcineurin, Drp1, and SP is higher than in normal skin., Conclusion: Human mast cell degranulation requires mitochondrial dynamics, also implicated in AD., (Copyright © 2011 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Objective: To investigate the mechanism of airway neurogenic inflammation by studying the expression of inducible nitric oxide synthase (iNOS) and substance P in C(7)-T(5) dorsal root ganglion sensory neuron cells induced by nerve growth factor (NGF) and the role of interferon regulatory factor-1 (IRF-1)., Methods: The dorsal root ganglia neuron (DRGn) cells were primary cultured, and then stimulated with or without NGF or NGF+interferon (IFN)-γ. Subsequently the DRGn cells were transinfected with or without green fluorescent protein (GFP)-IRF-1-vshRNA, and then stimulated with or without NGF. The expressions of iNOS and substance P were detected by real-time PCR., Results: NGF induced the mRNA expression of iNOS and substance P in dorsal root ganglion sensory neuron cells, and IFN-γ increased NGF-induced iNOS mRNA expression. The expressions of iNOS and substance P in sensory neuron cells were decreased significantly at the mRNA level after IRF-1 was blocked down by IRF-1-vshRNA transinfection., Conclusion: NGF is involved in the airway neurogenic inflammation by prompting the expression of iNOS and substance P through transcription factor IRF-1 in airway sensory neuron cells. IRF-1 may be a therapeutic target for airway neurogenic inflammation.

Transient receptor potential vanilloid 1 (TRPV1), neurokinin 1 (NK1) receptor and substance P (SP) immunoreactivity (-ir) and mRNA in the rat lumbosacral spinal cord and urinary bladder were measured 24h after s.c. injection of the vanilloids, capsaicin (50mg/kg) and resiniferatoxin (RTX, 100μg/kg), or vehicle (10% ethanol/10% Tween 80/saline). In the spinal cord, capsaicin significantly reduced TRPV1 and SP-ir (40-45%) in laminae I/II compared to controls, while RTX produced decreases of ~35%. NK1-ir in the spinal cord was unaffected by both vanilloid treatments. In the bladder, SP-ir was reduced in urothelial cells of some capsaicin- and RTX-treated rats, while SP-ir in the suburothelium and muscularis was significantly reduced by RTX. A significant increase in NK1-ir was observed in the urothelium and muscularis after capsaicin administration. Capsaicin significantly increased SP mRNA in the spinal cord, and TRPV1 and SP mRNA in the bladder, whereas RTX increased TRPV1, SP and NK1 mRNA in the spinal cord, and TRPV1 and SP mRNA in the bladder. These data suggest that stimulation of TRPV1 by low dose vanilloid administration can rapidly (within 24h) alter both transcription and translation of TRPV1 channels, SP and NK1 receptors in the rat urinary bladder and spinal cord., (2011 Elsevier B.V. All rights reserved.)

Objective: To evaluate the gene and protein expression of nerve growth factor (NGF) and substance P (SP) in the bladder 8 weeks after diabetes induction and investigate the pathogenesis of diabetic cystopathy., Methods: Thirty Wistar rats were divided into three groups: control (n = 10), streptozotocin-induced diabetic group (n = 10) and Pygeum africanum (P. africanum) group (n = 10; diabetic rats were given P. africanum (100 mg/kg/day)). Eight weeks later, the bladders were dissected. We measured the expression of NGF and SP in the bladders using RT-PCR, ELISA and immunohistochemistry., Results: We found a significantly reduced expression of NGF in the bladders from the diabetic group compared with the control. Immunohistochemical studies showed a statistically significant reduction of SP in the bladders from the diabetic group compared with the control (P < 0.05). Expression of NGF was greatly increased in the P. africanum group compared with that of the diabetic group. Immunohistochemical studies showed an increased level of SP in the bladders from the P. africanum group compared with the control (P < 0.05)., Conclusions: Our findings indicated that the decrease in NGF and SP may be a contributory factor in diabetic cystopathy. In addition, P. africanum could significantly upregulate the expression of NGF and SP in diabetic rats.

The neuropeptide substance-P (SP) is expressed from the TAC1 gene in sensory neurones where it acts as a key modulator of neurogenic inflammation. The promoter of TAC1 (TAC1prom) plays a central role in the regulation of the TAC1 gene but requires the presence of a second regulatory element; ECR2, to support TAC1 expression in sensory neurones and to respond appropriately to signalling pathways such as MAPkinases and noxious induction by capsaicin. We examined whether the effect of capsaicin on ECR2-TAC1prom activity in larger diameter neurones was cell autonomous or non- cell autonomous. We demonstrate that TRPV1 is not expressed in all the same cells as SP following capsaicin induction suggesting the presence of a non-cell autonomous mechanism for TAC1 up-regulation following capsaicin induction. In addition, we demonstrate that induction of SP and ECR1-TAC1prom activity in these larger diameter neurones can be induced by potassium depolarisation suggesting that, in addition to capsaicin induction, transgene activity may be modulated by voltage gated calcium channels. Furthermore, we show that NK1 is expressed in all SP- expressing cells after capsaicin induction and that an agonist of NK1 can activate both SP and the transgene in larger diameter neurones. These observations suggest the presence of an autocrine loop that controls the expression of the TAC1 promoter in sensory neurones. In contrast, induction of the TAC1 promoter by LPS was not dependent on ECR2 and did not occur in large diameter neurones. These studies demonstrate the diversity of mechanisms modulating the activity of the TAC1 promoter and provide novel directions for the development of new anti-inflammatory therapies.

It has been hypothesised that substance P (SP) may be produced by primary fibroblastic tendon cells (tenocytes), and that this production, together with the widespread distribution of the neurokinin-1 receptor (NK-1 R) in tendon tissue, could play an important role in the development of tendinopathy, a condition of chronic tendon pain and thickening. The aim of this study was to examine the possibility of endogenous SP production and the expression of NK-1 R by human tenocytes. Because tendinopathy is related to overload, and because the predominant tissue pathology (tendinosis) underlying early tendinopathy is characterized by tenocyte hypercellularity, the production of SP in response to loading/strain and the effects of exogenously administered SP on tenocyte proliferation were also studied. A cell culture model of primary human tendon cells was used. The vast majority of tendon cells were immunopositive for the tenocyte/fibroblast markers tenomodulin and vimentin, and immunocytochemical counterstaining revealed that positive immunoreactions for SP and NK-1 R were seen in a majority of these cells. Gene expression analyses showed that mechanical loading (strain) of tendon cell cultures using the FlexCell© technique significantly increased the mRNA levels of SP, whereas the expression of NK-1 R mRNA decreased in loaded as compared to unloaded tendon cells. Reduced NK-1 R protein was also observed, using Western blot, after exogenously administered SP at a concentration of 10⁻⁷ M. SP exposure furthermore resulted in increased cell metabolism, increased cell viability, and increased cell proliferation, all of which were found to be specifically mediated via the NK-1 R; this in turn involving a common mitogenic cell signalling pathway, namely phosphorylation of ERK1/2. This study indicates that SP, produced by tenocytes in response to mechanical loading, may regulate proliferation through an autocrine loop involving the NK-1 R.

Histological characterization of spinal fusions in Atlantic salmon (Salmo salar) has demonstrated shape alterations of vertebral body endplates, a reduced intervertebral space, and replacement of intervertebral cells by ectopic bone. However, the significance of the notochord during the fusion process has not been addressed. We have therefore investigated structural and cellular events in the notochord during the development of vertebral fusions. In order to induce vertebral fusions, Atlantic salmon were exposed to elevated temperatures from fertilization until they attained a size of 15g. Based on results from radiography, intermediate and terminal stages of the fusion process were investigated by immunohistochemistry and real-time quantitative polymerase chain reaction. Examination of structural extracellular matrix proteins such as Perlecan, Aggrecan, Elastin, and Laminin revealed reduced activity and reorganization at early stages in the pathology. Staining for elastic fibers visualized a thinner elastic membrane surrounding the notochord of developing fusions, and immunohistochemistry for Perlecan showed that the notochordal sheath was stretched during fusion. These findings in the outer notochord correlated with the loss of Aggrecan- and Substance-P-positive signals and the further loss of vacuoles from the chordocytes in the central notochord. At more progressed stages of fusion, chordocytes condensed, and the expression of Aggrecan and Substance P reappeared. The hyperdense regions seem to be of importance for the formation of notochordal tissue into bone. Thus, the remodeling of notochord integrity by reduced elasticity, structural alterations, and cellular changes is probably involved in the development of vertebral fusions.

Substance P (SP) has a widespread distribution in the whole body. After binding to the neurokinin-1 (NK-1) receptor, SP regulates biological functions related to cancer: tumor cell proliferation (favoring tumor growth), angiogenesis, and migration of the tumor cells for invasion and metastasis. SP also exerts an antiapoptotic effect. The peptide is secreted from primary tumors and from peripheral nerves, and reaches the whole body through the blood stream. NK-1 receptors are overexpressed in tumors (cancer cells express more NK-1 receptors than normal cells). By contrast, after binding to NK-1 receptors, the NK-1 receptor antagonists specifically inhibit tumor cell proliferation (tumor cells die by apoptosis), angiogenesis and the migration of the tumor cells. Thus, 1) the SP/NK-1 receptor system plays an important role in the development of cancer, angiogenesis, and metastasis; 2) a common mechanism for cancer cell proliferation mediated by the SP/NK-1 receptor system occurs; 3) NK-1 receptor antagonists act as a broad-spectrum antitumoral agent; 4) the NK-1 receptor could be a new promising target in the treatment of cancer; 5) NK-1 receptor antagonists could improve cancer treatment--the development of antagonist molecules of the NK-1 receptor represents an important opportunity for exploiting these molecules as novel therapeutic agents.

Clinical data provide evidence of high level of co-morbidity among genitourinary and gastrointestinal disorders characterized by chronic pelvic pain. The objective of this study was to test the hypothesis that colonic inflammation can impact the function of the urinary bladder via activation of TRPV1 signaling pathways followed by alterations in gene and protein expression of substance P (SP) and calcitonin gene-related peptide (CGRP) in sensory neurons and in the bladder. Inflammation was induced by intracolonic instillation of trinitrobenzene sulfonic acid (TNBS, 12.5mg/kg), and desensitization of TRPV1 receptors was evoked by intracolonic resiniferatoxin (RTX, 10(-)(7)M). mRNA and protein concentrations of CGRP and SP were measured at 3, 5 and 30 days. RTX instillation in the colon caused 3-fold up-regulation of SP mRNA in the urinary bladder at day 5 (n=7, p ≤ 0.05) followed by 35-fold increase at day 30 (n=5, p ≤ 0.05). Likewise, TNBS colitis triggered 15.8-fold up-regulation of SP mRNA 1 month after TNBS (n=5, p ≤ 0.05). Desensitization of colonic TRPV1 receptors prior to TNBS abolished SP increase in the urinary bladder. RTX led to 4.3-fold increase of CGRP mRNA at day 5 (n=7, p ≤ 0.05 to control) in the bladder followed by 28-fold increase at day 30 post-RTX (n=4, p ≤ 0.05). Colitis did not alter CGRP concentration during acute phase; however, at day 30 mRNA level was increased by 17.8 ± 6.9-fold (n=5, p ≤ 0.05) in parallel with 4-fold increase in CGRP protein (n=5, p ≤ 0.01) in the detrusor. Protein concentration of CGRP in the spinal cord was diminished by 45-65% (p ≤ 0.05) during colitis. RTX pretreatment did not affect CGRP concentration in the urinary bladder; however, it caused a reduction in CGRP release from lumbosacral DRG neurons during acute phase (3 and 5 days post-TNBS). Our results clearly demonstrate that colonic inflammation triggers the release of pro-inflammatory neuropeptides SP and CGRP in the urinary bladder via activation of TRPV1 signaling mechanisms enunciating the neurogenic nature of pelvic organ cross-sensitization., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Prostaglandin E2 (PGE2) is a well known pain and pro-inflammatory mediator abundantly produced in inflamed tissue. It causes pain by directly exciting nociceptive primary sensory neurons (nociceptors) and indirectly stimulating the release of pain-related peptide substance P (SP) and calcitonin gene-related peptide (CGRP). In an ex vivo culture of sensory ganglion explants, we tested the hypothesis that PGE2 could induce the synthesis of SP and CGRP in nociceptors. A stabilized PGE2 analog, 16,16-dimethyl PGE2, in a concentration- and time-dependent manner, significantly increased mRNA and peptide levels of SP and CGRP. The agonists of EP1 and EP4 receptors also significantly increased SP and CGRP levels. Moreover, 16,16-dimethyl PGE2-induced SP and CGRP were blocked by EP1 and EP4 antagonists as well as the inhibitors of both protein kinase A and protein kinase C. Nerve growth factor was partially involved in PGE2-induced SP and CGRP synthesis. Taken together, these results indicate that PGE2 contributes to the synthesis of SP and CGRP in nociceptors, an event mediated by EP1 and EP4 receptors, nerve growth factor and protein kinase A and protein kinase C signalling pathways. We thus conclude that facilitating the synthesis of pain-related peptides in nociceptors is a novel mechanism underlying the role of PGE2 in nociception and chronic pain states., (© 2010 The Author. Journal Compilation © 2010 International Society for Neurochemistry.)