Expression Of Neurokinin Research Articles

P–597 Placental expression of neurokinin B and its receptor NK3R is increased in women with polycystic ovary syndrome: results of a preliminary study

Abstract Study question Is the placental expression of neurokinin B (NKB) and its receptors NK1R, NK2R and NK3R affected by the polycystic ovary syndrome (PCOS)? Summary answer: The placental expression of NKB and NK3R is increased in PCOS, while the expression of NK1R and NK2R is not affected. What is known already: Women with PCOS are at increased risk of pregnancy complications and poor pregnancy outcomes. Defective placentation is among the proposed mechanisms involved. Altered NKB placental expression has been associated with several conditions characterized by placental dysfunction, such as pre-eclampsia and intra-uterine growth retardation. To our knowledge, the expression of NKB and its receptors has not been studied in placental tissue of women with PCOS. Study design, size, duration This was a single-center, prospective, case-control study. Women with PCOS according to the Rotterdam criteria (cases) and healthy pregnant women (controls) were enrolled at first prenatal visit and followed until delivery. Only women with spontaneous conception and singleton, uncomplicated, term pregnancies (10 PCOS and 10 controls) were included in the final analysis. All participants provided informed consent. Participants/materials, setting, methods At delivery, placental specimens were collected and immediately submerged in RNAlater solution. Samples were stored at –20oC until analysis. The mRNA expression of NKB, NK1R, NK2R and NK3R was quantified by real-time PCR (RT-PCR). The relative mRNA expression was estimated by the ΔΔCT method, using β-actin as reference (housekeeping gene). Statistical analysis was performed using SPSS 25.0, and the level of statistical significance was set at 0.05 (two-sided). Main results and the role of chance The placental mRNA expression of NKB and NK3R was significantly higher in PCOS women versus controls (2.4-fold, p < 0.05 for NKB and 7-fold, p < 0.05 for NK3R). No significant alterations were observed in the mRNA expression of NK1R and NK2R between the two groups. There was no statistically significant difference regarding age, BMI, caesarian section frequency, offspring sex and birth weight between women with PCOS and controls. The placental expression of NKB and its receptors was correlated neither with maternal age and BMI, nor with offspring birth weight. Limitations, reasons for caution The main limitation of this study is the small sample size. Expanding the number of participants is the necessary next step, in order to corroborate our preliminary findings. Furthermore, correlations between the placental expression of NKB, NK1R, NK2R, NK3R and maternal sex steroids, glucose and insulin levels should be sought. Wider implications of the findings: The present study is the first to demonstrate increased placental expression of NKB and its receptor NK3R in women with PCOS. These findings support a potential role for NKB as a mediator of placental alterations characterizing PCOS. Trial registration number Not applicable

A Review of Studies on the Use of New Non-Gabapentinoid Anticonvulsants and Muscle Relaxants in the Control of Acute Postoperative Pain

Postoperative pain management in any type of surgery is an important issue for patients. The pain relief contributes to decreased hospital costs, shorter hospital stay, and elevated patient consent. Correspondingly, in this review article we assessed the postoperative pain management efficacy of new non-gabapentinoid anticonvulsants and muscle relaxant drugs in pre-clinical and clinical studies. The scientific databases, including PubMed, Embase, Scopus, and Google Scholar, were searched using relevant keywords: surgery, postoperative pain, analgesia, non-gabapentinoid anticonvulsants, and muscle relaxants. New anticonvulsants reduce postoperative pain, length of stay at postoperative anesthesia care unit, and analgesic requirement after surgeries. Their mechanisms include the inhibition of glutamate release, blocking the N-type calcium channels of afferent neurons, inhibition of supraspinal 5-hydroxytryptamine-3 receptor, and prevention of cyclin-dependent kinase 5-mediated collapsin response mediator protein 2 phosphorylation. Also, muscle relaxants show the same effects by downregulation the Cacna1a, Cacna1b, and Runt-related transcription factor 1 genes of dorsal root ganglia, inhibition the release of glutamate, aspartate or substance P from the terminals of primary afferent C and Aδ fibers, expression of Neurokinin 1 receptor in the spinal dorsal horn, agonistic effect on α2-adrenoceptor, and stimulation of acetylcholine release in the spinal cord. With respect to the concerns regarding opioid abuse, muscle relaxants and non-gabapentinoid anticonvulsant drugs can be regarded as a safe option for postoperative pain control.

Placental expression of neurokinin B and its receptor NK3R is increased in women with polycystic ovary syndrome: results of a preliminary study

Placental expression of neurokinin B and its receptor NK3R is increased in women with polycystic ovary syndrome: results of a preliminary study

MiR-206 Promotes Cancer Progression by Targeting Full-Length Neurokinin-1 Receptor in Breast Cancer.

Substance P plays a pivotal role in human cancer development and progression by binding to its receptor, neurokinin-1. Neurokinin-1 has 2 isoforms: full-length neurokinin-1 and truncated neurokinin-1, the latter lacking the cytoplasmic terminal 96-amino acid residues of the full-length protein. We have identified 3 candidate miR-206 target sites within the 3′-untranslated region of the full-length neurokinin-1 gene from bioinformatics database searches. In the present study, real-time quantitative polymerase chain reaction was performed to quantify the expression of miR-206, and the expression of neurokinin-1 and full-length neurokinin-1 was detected by immunohistochemistry in 82 clinical cases of breast cancer and paired adjacent normal tissues. The miR-206 target gene was demonstrated by using a dual-luciferase reporter assay, quantitative real-time polymerase chain reaction, and Western blotting. Transwell migration and invasion, colony formation, and proliferation assays were performed to evaluate the effects of miR-206 expression on various aspects of breast cancer cell behavior in vitro. We showed that miR-206 expression is upregulated in breast cancer cell lines and breast cancer tissues when compared to that in adjacent normal tissues, and full-length neurokinin-1 expression inversely correlates with Tumor Lymph Node Metastasis (TNM) stage and lymph node metastasis. Western blotting, quantitative real-time polymerase chain reaction, and dual-luciferase reporter assays demonstrated that miR-206 binds the 3′-untranslated region of full-length neurokinin-1 messenger RNA, regulating protein expression. We showed that the overexpression of miR-206 promotes breast cancer cell invasion, migration, proliferation, and colony formation in vitro. The present study furthers the current understanding of the mechanisms underlying breast cancer pathogenesis and may be useful for the development of novel targeted therapies.

Effects of allogeneic skin fibroblasts on promoting wound healing of diabetic mice and the mechanism

Effects of allogeneic skin fibroblasts on promoting wound healing of diabetic mice and the mechanism

Energy balance affects pulsatile secretion of luteinizing hormone from the adenohypophesis and expression of neurokinin B in the hypothalamus of ovariectomized gilts.

The pubertal transition of gonadotropin secretion in pigs is metabolically gated. Kisspeptin (KISS1) and neurokinin B (NKB) are coexpressed in neurons within the arcuate nucleus of the hypothalamus (ARC) and are thought to play an important role in the integration of nutrition and metabolic state with the reproductive neuroendocrine axis. The hypothesis that circulating concentrations of luteinizing hormone (LH) and expression of KISS1 and tachykinin 3(TAC3, encodes NKB) in the ARC of female pigs are reduced with negative energy balance was tested using ovariectomized, prepubertal gilts fed to either gain or lose body weight. Restricted feeding of ovariectomized gilts caused a rapid and sustained metabolic response characterized by reduced concentrations of plasma urea nitrogen, insulin, leptin, and insulin-like growth factor-1 and elevated concentrations of free fatty acids. The secretory pattern of LH shifted from one of low amplitude to one of high amplitude, which caused overall circulating concentrations of LH to be greater in restricted gilts. Nutrient-restricted gilts had greater expression of follicle-stimulating hormone and gonadotropin-releasing hormone receptor, but not LH in the anterior pituitary gland. Expression of KISS1 in the ARC was not affected by dietary treatment, but expression of TAC3 was greater in restricted gilts. These data are consistent with the idea that hypothalamic expression of KISS1 is correlated with the number of LH pulse in pig, and further indicate that amplitude of LH pulses may be regulated by NKB in the gilt.

Oxytocin Intranasal Administration Affects Neural Networks Upstream of GNRH Neurons.

The last decade has witnessed a surge in studies on the clinical applications of intranasal oxytocin as a method of enhancing social interaction. However, the molecular and cellular mechanisms underlying its function are not completely understood. Since oxytocin is involved in the regulation of hypothalamic-pituitary-gonadal axis by affecting the gonadotropin-releasing hormone (GNRH) system, the present study addressed whether intranasal application of oxytocin has a role in affecting GNRH expression in the male rat hypothalamus. In addition, we assessed expression of two excitatory (kisspeptin and neurokinin B) and two inhibitory (dynorphin and RFamide-related peptide-3) neuropeptides upstream of GNRH neurons as a possible route to relay oxytocin information. Here, adult male rats received 20, 40, or 80μg oxytocin intranasally once a day for 10 consecutive days, and then, the posterior (PH) and anterior hypothalamus (AH) dissected for evaluation of target genes. Using qRT-PCR, we found that oxytocin treatment increased Gnrh mRNA levels in both the PH and AH. In addition, oxytocin at its highest dose increased kisspeptin expression in the AH by around 400%, whereas treatments, dose dependently decreased kisspeptin mRNA in the PH. The expression of neurokinin B was increased from the basal levels following the intervention. Furthermore, although intranasal-applied oxytocin decreased hypothalamic RFamide-related peptide-3 mRNA level, the dynorphin mRNA was not affected. These observations are consistent with the hypothesis that applications of intranasal oxytocin can affect the GNRH system.

Ascending projections of nociceptive neurons from trigeminal subnucleus caudalis: A population approach

Second-order neurons in trigeminal subnucleus caudalis (Vc) and upper cervical spinal cord (C1) are critical for craniofacial pain processing and project rostrally to terminate in: ventral posteromedial thalamic nucleus (VPM), medial thalamic nuclei (MTN) and parabrachial nuclei (PBN). The contribution of each region to trigeminal nociception was assessed by the number of phosphorylated extracellular signal-regulated kinase-immunoreactive (pERK-IR) neurons co-labeled with fluorogold (FG). The phenotype of pERK-IR neurons was further defined by the expression of neurokinin 1 receptor (NK1).The retrograde tracer FG was injected into VPM, MTN or PBN of the right hemisphere and after seven days, capsaicin was injected into the left upper lip in male rats. Nearly all pERK-IR neurons were found in superficial laminae of Vc-C1 ipsilateral to the capsaicin injection. Nearly all VPM and MTN FG-labeled neurons in Vc-C1 were found contralateral to the injection site, whereas FG-labeled neurons were found bilaterally after PBN injection. The percentage of FG-pERK-NK1-IR neurons was significantly greater (>10%) for PBN projection neurons than for VPM and MTN projection neurons (<3%). pERK-NK1-IR VPM projection neurons were found mainly in the middle-Vc, while pERK-NK1-immunoreactive MTN or PBN projection neurons were found in the middle-Vc and caudal Vc-C1.These results suggest that a significant percentage of capsaicin-responsive neurons in superficial laminae of Vc-C1 project directly to PBN, while neurons that project to VPM and MTN are subject to greater modulation by pERK-IR local interneurons. Furthermore, the rostrocaudal distribution differences of FG-pERK-NK1-IR neurons in Vc-C1 may reflect functional differences between these projection areas regarding craniofacial pain.

Decreased Brain Neurokinin-1 Receptor Availability in Chronic Tennis Elbow.

Substance P is released in painful and inflammatory conditions, affecting both peripheral processes and the central nervous system neurokinin 1 (NK1) receptor. There is a paucity of data on human brain alterations in NK1 expression, how this system may be affected by treatment, and interactions between central and peripheral tissue alterations. Ten subjects with chronic tennis elbow (lateral epicondylosis) were selected out of a larger (n = 120) randomized controlled trial evaluating graded exercise as a treatment for chronic tennis elbow (lateral epicondylosis). These ten subjects were examined by positron emission tomography (PET) with the NK1-specific radioligand 11C-GR205171 before, and eight patients were followed up after treatment with graded exercise. Brain binding in the ten patients before treatment, reflecting NK1-receptor availability (NK1-RA), was compared to that of 18 healthy subjects and, longitudinally, to the eight of the original ten patients that agreed to a second PET examination after treatment. Before treatment, patients had significantly lower NK1-RA in the insula, vmPFC, postcentral gyrus, anterior cingulate, caudate, putamen, amygdala and the midbrain but not the thalamus and cerebellum, with the largest difference in the insula contralateral to the injured elbow. No significant correlations between brain NK1-RA and pain, functional severity, or peripheral NK1-RA in the affected limb were observed. In the eight patients examined after treatment, pain ratings decreased in everyone, but there were no significant changes in NK1-RA. These findings indicate a role for the substance P (SP) / NK1 receptor system in musculoskeletal pain and tissue healing. As neither clinical parameters nor successful treatment response was reflected in brain NK1-RA after treatment, this may reflect the diverse function of the SP/NK1 system in CNS and peripheral tissue, or a change too small or slow to capture over the three-month treatment.

Substance P Stimulates Endothelin 1 Secretion via Endothelin-Converting Enzyme 1 and Promotes Melanogenesis in Human Melanocytes

Substance P (SP) is a well-known neuropeptide implicated in the wound-healing process. The wound occasionally causes a pigmented scar. In the present study, we examined whether increased levels of SP affected melanogenesis. When human melanocytes were treated with SP, the melanin content increased and the pigmentation process accelerated in a dose-dependent manner. In addition to melanogenesis-related genes, the expression of neurokinin 1 receptor, endothelin 1 (EDN1), and EDN receptor type B (EDNRB) also increased at both the messenger RNA and protein levels. Interestingly, secreted EDN1 was observed in the melanocyte culture medium, and this phenomenon was significantly enhanced by SP treatment. Through knockdown experiments using small interfering RNAs (siRNAs), we confirmed that endothelin-converting enzyme 1 (ECE1), EDN1, and EDNRB were involved in SP-induced pigmentation and found that EDN1 secretion was affected by ECE1 and EDN1 siRNAs, but not by EDNRB siRNA. These findings indicate that ECE1 is essential for EDN1 secretion in melanocytes and that EDNRB functions downstream of secreted EDN1 to increase the cAMP levels and activate the melanogenesis-related phosphorylation cascade. This study provides in vitro evidence for a melanogenic function of SP in the skin and suggests that the SP-related signal is a potent target for regulating stress- or wound-induced pigmentation.

Validation of Reference Genes for mRNA Quantification in Adjuvant Arthritis

Real time quantitative PCR (RT-qPCR) requires a method to normalize the expression of target genes against an en- dogenous reference gene. It is known that commonly used housekeeping genes (HKGs) vary tremendously in inflam- matory conditions; however information about the stability and expression of HKGs in chronic inflammatory joint dis- ease such as rheumatoid arthritis (RA) is scarce. The expressional stability of 10 commonly used HKGs was analyzed in the neuronal (spinal cord, dorsal root ganglia) and in the musculoskeletal tissues (tendon, muscle, epiphysis, capsule, periosteum and ankle joint) using RT-qPCR in the rat model of RA. In individual tissues, suitable HKGs were selected by | △Ct| (│Ct control-Ct arthritis│) and further analyzed by using software programs; geNorm and normfinder. We found hypoxanthine-guanine phosphoribosyl tranferase (HPRT) as the most stable gene except ankle joint while glyce-raldehyde-3-phosphate dehydrogenase (GAPDH) was found as the least stable gene in musculoskeletal tissues. In in-flamed ankle joint where no reference gene was found to be stably expressed, an inflammatory cell marker CD3 was used to normalize peptidylprolyl isomerase B (PPIB), the most homogenous HKG identified among the 10 HKGs. The normalized PPIB was then used to analyze the gene expression of neurokinin 1 (NK1), receptor of substance P, a potent pro-inflammatory mediator. We observed a 3.5 fold increase (p = 0.009) in NK1 expression in inflamed ankle joint compared to control. Our results indicate that reference genes stability should be evaluated before using them as refer- ence during inflammatory conditions. In tissues with intense inflammatory cell infiltration, an inflammatory cell marker should be used to normalize the selected reference gene to avoid erroneous results.

Expression of neurokinin B in hypertensive disorder complicating pregnancy

Objective To investigate the relationship between the genesis of hypertensive disorder complicating pregnancy and neurokinin B (NKB).Methods The serum NKB levels of 229 women including non-pregnant women (n = 62),normal pregnant women (n = 80) and pregnant women with preeclampsia (n=37) or gestational hypertension (n= 50) were examined by enzyme linked immunosorbent assay.The umbilical blood levels of NKB and the NKB mRNA expression in the pregnant women were also tested by fluorescent quantitative reverse transcription-polymerase chain reaction technique.ANOVA and paired t-test were applied.Results (1)The maternal plasma NKB levels of normal pregnant women,patients with gestational hypertension or preeclampsia were significantly higher than that of non-pregnant women,respectively [(28.2±6.6)μg/L,(31.5±5.2)μg/L,(70.5±8.9)μg/L vs(3.2±1.8)μg/L,P＜ 0.01].In preeclampsia,gestational hypertension and normal pregnancy group,the NKB level in umbilical blood [(121.4±9.3)μg/L,(60.5±7.2)μg/L,(40.8±6.3)μg/L] were significantly higher than that of maternal plasma(P＜0.01).(2) The maternal plasma NKB level in gestational hypertension group was higher than that in normal pregnancy group,while the difference had no statistical significance (P＞0.05).The maternal plasma NKB level in preeclampsia group was higher than that in gestational hypertension group and normal pregnancy group (P＜0.01).Forty-eight hours after delivery,the maternal plasma NKB levels significantly decreased in the normal pregnant,gestational hypertension and preeclampsia group [ (14.1±4.2) μg/L,(16.4±3.8) μg/L,(25.4±5.2) μg/L],compared to the values before delivery (P＜0.01).(3) The expression of NKB mRNA in placenta of preeclampsia group was significantly higher than that of gestational hypertension group and normal pregnancy group [(3.8±0.6) × 10-3 vs(1.7±0.4) × 10-3 and (1.6±0.3) × 10-3,P＜0.01].No statistical difference was found in the expression of NKB mRNA in placenta between gestational hypertension group and normal pregnancy group (P＞0.05).Conclusions Elevated NKB level in placenta and maternal or umbilical blood might play an important role in the development of hypertensive disorder complicating pregnancy. Key words: Hypertension,pregnancy-induced; Neurokinin B; Fetal blood; Placenta

Effects of a High-Salt Diet on TRPV-1-Dependent Renal Nerve Activity in Dahl Salt-Sensitive Rats

Objective: To test the hypothesis that transient receptor potential vanilloid type 1 channel (TRPV1)-mediated increases in afferent renal nerve activity (ARNA) and release of substance P (SP) and calcitonin gene-related peptide (CGRP) from the renal pelvis are suppressed in Dahl salt-sensitive (DS), but not -resistant (DR), rats fed a high-salt (HS) diet. Methods and Results: Male DS and DR rats were given a HS or low-salt (LS) diet for 3 weeks. Perfusion of capsaicin (CAP, 10^–6M), a selective TRPV1 agonist, into the left renal pelvis increased ipsilateral ARNA in all groups, but with a smaller magnitude in DS-HS compared to other groups. CAP increased contralateral urine flow in all groups except DS-HS rats. CAP-induced release of SP and CGRP from the renal pelvis was less in DS-HS compared to other groups. Western blot showed that TRPV1 expression in the kidney decreased while expression of neurokinin 1 receptors increased in DS-HS compared to other groups. Conclusion: TRPV1-mediated increases in ARNA and release of SP and CGRP in the renal pelvis are impaired in DS rats fed a HS diet, which can likely be attributed to suppressed TRPV1 expression in the kidney and contributes to increased salt sensitivity.

Increased neuropeptides in spinal cord with performance of a highly repetitive, forceful task are attenuated with anti‐inflammatory and ergonomic interventions

In spite of the abundant epidemiological evidence linking exposure to repetitive and/or forceful motion with repetitive strain injuries, effective interventions are still lacking. This study examined the effectiveness of 2 interventions (ergonomic modification and ibuprofen) in reducing nociceptive neuropeptides and inflammation after a highly repetitive, forceful reaching/grasping (HRHF) task. Rats were randomly assigned to 4 groups (ergonomic intervention, medicated, HRHF, or controls; 5–8/group). Controls learned, but did not perform the regimen. HRHF rats reach/grasp every 15 s at 70% of max grip strength (2hr/d, 3d/wk, 6–12 wks). Ergonomic rats performed the HRHF task for 4 wks, then transfer to a low repetition‐negligible force (LRNF) task (reach/grasp every 30 s, 20% of max). Medicated rats receive oral ibuprofen after HRHF task wk 4 and continued the task. Motor function was monitored weekly. Immunochemistry was used to analyze serum cytokines, nerve macrophages, and spinal Substance P (SP) and Neurokinin‐1 (NK‐1). In serum, ibuprofen reduced IL‐1a, interferon‐gamma, MIP2 and MIP3a, while ergonomic intervention reduced IL‐1a and MIP2. Nerve macrophages and spinal SP and NK‐1 expression were reduced by both interventions. These data suggest that a lowered work demand is as effective as ibuprofen in reducing inflammation and neuropeptides induced by repetitive, forceful tasks. NIAMS‐AEBarr

The effect of high cervical spinal cord stimulation on the expression of SP, NK-1 and TRPV1 mRNAs during cardiac ischemia in rat

Spinal cord stimulation (SCS) is used to reduce angina that accompanies cardiac ischemia, but little is known about the molecular mechanisms mediating this effect. We studied the expression of SP, neurokinin-1 (NK-1) receptor, and transient receptor potential vanilloid type 1 (TRPV1) mRNA in the rat spinal cord at thoracic 4 (T4), cervical 2 (C2) and caudal brain stem by RT-PCR during intermittent occlusion of the left anterior descending coronary artery (CoAO), during sustained SCS by itself at the C2 spinal segment, and during sustained SCS plus intermittent CoAO. Only SP mRNA was increased significantly in T4 and brainstem during CoAO, while SCS decreased the mRNA levels of SP, NK-1 and TRPV1 significantly in T4 and the brainstem. SCS attenuated the increase of SP and TRPV1 mRNA levels at T4 level induced by intermittent CoAO when the stimulation was applied prior to the initiation of the cardiac ischemia. These results support the role for SP as a putative neurotransmitter for the myocardial ischemia-sensitive afferent neuron signal to the spinal level. They suggest that modification of the ischemic cardiac nociceptive afferent signal by SCS involves a change in SP and TRPV1 expression.

Nuclear Factor-κB Is Central to the Expression of Truncated Neurokinin-1 Receptor in Breast Cancer: Implication for Breast Cancer Cell Quiescence within Bone Marrow Stroma

Breast cancer is a leading cause of mortality among women in the United States. Tac1 and neurokinin-1 (NK1) are involved in autocrine stimulation of breast cancer cells (BCCs). The single NK1 gene produces full-length (NK1-FL) and truncated (NK1-Tr) forms. NK1-Tr mediates malignancy in breast cells. We now report a critical role for nuclear factor-kappaB (NF-kappaB) in the expression of NK1-Tr, but not NK1-FL, in human BCCs. By Western and Northern blot analyses, NK1-FL and NK1-Tr were coexpressed in BCCs but were undetectable in nontumorigenic cells. Loss of repressive activity within the 5' flanking region of the NK1 partly accounts for constitutive expression of NK1 in BCCs but could not account for the presence of NK1-Tr. Transient transfections with dominant-negative and wild-type IkappaB show that activation of NF-kappaB is required for the expression of NK1-Tr. Tac1 gene was linked to the generation of NK1-Tr because its overexpression in BCCs led to the production of multiple cytokines that can activate NF-kappaB to mediate NK1-Tr expression. Studies with Tac1 knockdown BCCs and Tac1-expressing nontumorigenic breast cells verified a role for NF-kappaB in the expression of NK1-Tr. The quiescent phenotype of BCCs on contact with bone marrow stroma was partly explained by decreased NF-kappaB activation and undetectable NK1-Tr. In summary, this study shows a role for NF-kappaB in the expression of NK1-Tr in BCCs, which seems to be reversed by bone marrow stromal cells.

Hypoxia upregulates lung microvascular neurokinin-1 receptor expression

Subacute exposure to moderate hypoxia can promote pulmonary edema formation. The tachykinins, a family of proinflammatory neuropeptides, have been implicated in the pathogenesis of pulmonary edema in some settings, including the pulmonary vascular leak associated with exposure to hypoxia. The effects of hypoxia on tachykinin receptor and peptide expression in the lung, however, remain poorly understood. We hypothesized that subacute exposure to moderate hypoxia increases lung neurokinin-1 (NK-1) receptor expression as well as lung substance P levels. We tested this hypothesis by exposing weanling Sprague-Dawley rats to hypobaric hypoxia (barometric pressure 0.5 atm) for 0, 24, 48, or 72 h. Hypoxia led to time-dependent increases in lung NK-1 receptor mRNA expression and lung NK-1 receptor protein levels at 48 and 72 h of exposure (P < 0.05). Immunohistochemistry and in situ NK-1 receptor labeling with substance P-conjugated fluorescent nanocrystals demonstrated that hypoxia increased NK-1 expression primarily in the pulmonary microvasculature and in alveolar macrophages. Hypoxia also led to increases in lung substance P levels by 48 and 72 h (P < 0.05) but led to a decrease in preprotachykinin mRNA levels (P < 0.05). We conclude that subacute exposure to moderate hypoxia upregulates lung NK-1 receptor expression and lung substance P peptide levels primarily in the lung microvasculature. We speculate that this effect may contribute to the formation of pulmonary edema in the setting of regional or environmental hypoxia.

The expression of neurokinin-1 and preprotachykinin-1 in breast cancer cells depends on the relative degree of invasive and metastatic potential

Breast cancer has a predilection for metastasis to the bone marrow. The preprotachykinin-I (PPT-I) gene has a central role in the early migration of breast cancer cells into the bone marrow, making this organ a latent repository of the cancer cells. This study investigated whether the invasive and metastatic potential of breast cancer cells correlate with the expression of the PPT-I gene and the receptors for its peptides, neurokinin-1 (NK-1) and NK-2. The studies compared cells that are non-tumorigenic (MCF12A), low metastatic and invasive potential (MCF7), and sublines of MCF with increased invasive and metastatic potential (LCC1 and LCC2). LCC2, but not LCC1 is tamoxifen resistant. Quantitative RT-PCR showed increased expression of PPT-I, NK-1 and NK-2 mRNA LCC1 and LCC2. MCF7 required stimulation by phorbol ester for NK-1 induction. The levels of NK-2 mRNA were significantly increased in LCC2. Clonogenic assays with specific receptor antagonists showed a predominant role for NK-2 in the proliferation of both LCC1 and LCC2. While the growth rate of LCC1 and LCC2 were similar, the latter showed increased migration. Use of a nude mouse model confirmed higher metastatic potential of LCC2, including increased migration to regions of the endosteum. Overall, these studies show a correlation between three neuroendocrine-related genes: PPT-I, NK-1 and NK-2 and the metastatic potential of specific breast cancer cells. These cells provide a model for future studies on bone marrow metastasis.

Regulation of neurokinin-1 receptor messenger RNA expression in synovial fibroblasts of patients with rheumatoid arthritis

We examined whether soluble mediators regulate the expression of tachykinin receptor mRNAs in synovial fibroblasts of patients with rheumatoid arthritis (RA). mRNAs encoding long and short isomers of neurokinin 1 receptor (NK1R), and neurokinin 2 receptor (NK2R) were confirmed by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Level of long, but not the short, of NK1R mRNA was increased by treatment with 10-100 ng/ml basic fibroblast growth factor (bFGF) or 20 ng/ml tumor necrosis factor-alpha (TNF-alpha), but not with 1ng/ml interleukin 1beta (IL-1beta). TNF-alpha upregulated NK2R mRNA as well as long NK1R mRNA whereas bFGF had no effect on NK2R mRNA. Expression of neurokinin 3 receptor (NK3R) mRNA was not observed in RA fibroblasts, and its expression was not induced by bFGF and TNF-alpha. The basal and increased levels of long NK1R mRNA were inhibited by treatment with 20 microM SU5402, an inhibitor of the tyrosine kinase activity of FGF receptor 1 (FGFR1), or 10 ng/ml transforming growth factor-beta1 (TGF-beta1). SU5402 and TGF-beta1 had no effect on the basal level of short NK1R mRNA. Immunocytochemistry revealed the enhancement by bFGF of immunoreactive NK1Rs in the cells at 24 h after treatment. These results suggest that bFGF, TGF-beta1, and TNF-alpha in synovial tissue and fluid play a role in the regulation of long NK1R expression in synovial fibroblasts of RA patients. It appears that the pathway of downregulation by TGF-beta1 is more dominant in the long NK1R mRNA expression than that of upregulation by bFGF or TNF-alpha. Furthermore, the regulation of short NK1R mRNA expression seems to be performed via a different pathway from that of long isomer mRNA.

Autocrine proliferation of neuroblastoma cells is partly mediated through neurokinin receptors: relevance to bone marrow metastasis

Despite intensive therapy, approximately 60-80% of children who are diagnosed with metastatic neuroblastoma (NB) succumb to the disease. NB preferentially metastasizes to the bone marrow (BM). In the present study we used SY5Y and CHP212 (NB cell lines) to study the roles of the preprotachykinin-I (PPT-I) gene and the natural receptors for PPT-I peptides, neurokinin-1 (NK-1) and NK-2, in the biology of NB. PPT-I, NK-1 and NK-2 were constitutively expressed in the NB cells. Functional studies, with specific NK receptor antagonists, showed that PPT-I peptides mediate autocrine proliferation of the NB cells through both NK-1 and NK-2 receptors. Full-length and truncated NK-1 receptors were detected in NB cells. Since there is one copy of the NK-1 gene, we used NK-1-specific siRNA to suppress the expression of NK-1. The NK-1-deficient NB cells showed phenotypes consistent with cell differentiation. Suppression of NK-1 did not appear to cause cell death, as demonstrated by trypan blue exclusion and by undetectable active caspase. NK-1 suppression reduced the proliferation of the NB cells beginning by 10-fold at day 1 and reached a 10(5)-fold reduction by day 10. The NK-1 deficient cells did not proliferate when they were placed as cocultures with BM stroma, which suggests that NK-1 signaling is important for the survival of NB cells in the BM. The results show potential roles for NK receptors in the proliferation of NB.