Differential Influences of Endogenous and Exogenous Sensory Neuropeptides on the ATP Metabolism by Soluble Ectonucleotidases in the Murine Bladder Lamina Propria.

Bladder urothelium and suburothelium/lamina propria (LP) have prominent sensory and transducer functions with the active participation of afferent neurons and urothelium-derived purine mediators such as adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine (ADO). Effective concentrations of purines at receptor targets depend significantly on the extracellular degradation of ATP by ectonucleotidases (ENTDs). We recently reported the regulated release of soluble ENTDs (s-ENTDs) in the LP and the consequent degradation of ATP to ADP, AMP, and ADO. Afferent neurons in the LP can be activated by urothelial ATP and release peptides and other transmitters that can alter the activity of cells in their vicinity. Using a murine decentralized ex vivo detrusor-free bladder model, 1,N6-etheno-ATP (eATP) as substrate, and sensitive HPLC-FLD methodologies, we found that exogenous neuropeptides calcitonin gene-related peptide (CGRP), substance P (Sub P), neurokinin A (NKA), and pituitary adenylate cyclase-activating polypeptide [PACAP (1-38)] all increased the degradation of eATP by s-ENTDs that were released in the LP spontaneously and/or during bladder filling. Using antagonists of neuropeptide receptors, we observed that endogenous NKA did not modify the ATP hydrolysis by s-ENTDs, whereas endogenous Sub P increased both the constitutive and distention-induced release of s-ENTDs. In contrast, endogenous CGRP and PACAP (1-38) increased the distention-induced, but not the spontaneous, release of s-ENTDs. The present study puts forward the novel idea that interactions between peptidergic and purinergic signaling mechanisms in the LP have an impact on bladder excitability and functions by regulating the effective concentrations of adenine purines at effector cells in the LP.

Deciphering specificity and cross-reactivity in tachykinin NK1 and NK2 receptors.

The tachykinin receptors neurokinin 1 (NK1R) and neurokinin 2 (NK2R) are G protein-coupled receptors that bind preferentially to the natural peptide ligands substance P and neurokinin A, respectively, and have been targets for drug development. Despite sharing a common C-terminal sequence of Phe-X-Gly-Leu-Met-NH2 that helps direct biological function, the peptide ligands exhibit some degree of cross-reactivity toward each other's non-natural receptor. Here, we investigate the detailed structure-activity relationships of the ligand-bound receptor complexes that underlie both potent activation by the natural ligand and cross-reactivity. We find that the specificity and cross-reactivity of the peptide ligands can be explained by the interactions between the amino acids preceding the FxGLM consensus motif of the bound peptide ligand and two regions of the receptor: the ß-hairpin of the extracellular loop 2 (ECL2) and a N-terminal segment leading into transmembrane helix 1. Positively charged sidechains of the ECL2 (R177 of NK1R and K180 of NK2R) are seen to play a vital role in the interaction. The N-terminal positions 1 to 3 of the peptide ligand are entirely dispensable. Mutated and chimeric receptor and ligand constructs neatly swap around ligand specificity as expected, validating the structure-activity hypotheses presented. These findings will help in developing improved agonists or antagonists for NK1R and NK2R.

The Role of Substance P, Neurokinin A, Neuropeptide Y, and Cortisol in Assessing Neonatal Pain.

Introduction: Our aim was to investigate biomarkers of neonatal pain and their association with two pain scales. Methods: This prospective study included 54 full-term neonates. Levels of substance P (SubP), neurokinin A (NKA), neuropeptide Y (NPY), and cortisol were recorded and two pain scales (Premature Infant Pain Profile [PIPP] and Neonatal Infant Pain Scale [NIPS]) were used. Results: A statistically significant decrease in the levels of NPY (p = 0.02) and NKA (p = 0.03) was detected. A significant increase in NIPS scale (p < 0.001) and PIPP scale (p < 0.001) postpainful intervention was also detected. There was a positive correlation between cortisol and SubP (p = 0.01), NKA and NPY (p < 0.001) and between NIPS and PIPP (p < 0.001). A negative correlation was found for NPY with SubP (p = 0.004), cortisol (p = 0.02), NIPS (p = 0.001) and PIPP (p = 0.002). Conclusions: Novel biomarkers and pain scales may help in designing an objective tool for the quantification of neonatal pain in the everyday practice.

Colorectal and bladder prokinetic activity of [Lys5, MeLeu9, Nle10]-NKA(4-10) after intranasal or sublingual delivery in dogs.

The feasibility of eliciting defecation and urination after intranasal (IN) or sublingual (SL) delivery of a small peptide NK2 receptor agonist, [Lys5, MeLeu9, Nle10]-NKA(4-10), was examined using prototype formulations in dogs. In anesthetized animals, administration of 100 or 300 µg/kg IN or 2.0-6.7 mg/kg SL increased colorectal peak pressure and area under the curve. Peak bladder pressure was also increased at the same doses, and this was accompanied by highly efficient voiding at normal physiological bladder pressure. The onset of these effects was rapid (≤2.5 min), and the primary contractions lasted ∼25 min, returning to baseline in <60 min. Slight hypotension lasting a few minutes and causing <10% change from baseline was detected after higher doses and was statistically significant after only 100 µg/kg IN. In conscious dogs, there was a dose-related increase in voiding responses and reduction in the latency to urinate and defecate after 300 and 1000 µg/kg IN; emesis was also observed at these doses. SL administration of 6.7 mg/kg induced urination within 10 min, but not defecation or emesis. These findings support the feasibility of developing a convenient dosage form of small peptide NK2 receptor agonists as on-demand defecation or urination therapies.

IFN-γ-STAT1-mediated NK2R expression is involved in the induction of antitumor effector CD8+ T cells in vivo.

The induction of antitumor effector T cells in the tumor microenvironment is a crucial event for cancer immunotherapy. Neurokinin receptor 2 (NK2R), a G protein-coupled receptor for neurokinin A (NKA), regulates diverse physiological functions. However, the precise role of NKA-NK2R signaling in antitumor immunity is unclear. Here, we found that an IFN-γ-STAT1 cascade augmented NK2R expression in CD8+ T cells, and NK2R-mediated NKA signaling was involved in inducing antitumor effector T cells in vivo. The administration of a synthetic analog of double-stranded RNA, polyinosinic-polycytidylic acid (poly I:C), into a liver cancer mouse model induced type I and type II IFNs and significantly suppressed the tumorigenesis of Hepa1-6 liver cancer cells in a STAT1-dependent manner. The reduction in tumor growth was diminished by the depletion of CD8+ T cells. IFN-γ stimulation significantly induced NK2R and tachykinin precursor 1 (encodes NKA) gene expression in CD8+ T cells. NKA stimulation combined with anti-CD3 monoclonal antibody (mAb) treatment significantly augmented IFN-γ and granzyme B production by CD8+ T cells compared with the anti-CD3 mAb alone in vitro. ERK1/2 phosphorylation and IκBα degradation in activated CD8+ T cells were suppressed under NK2R deficiency. Finally, we confirmed that tumor growth was significantly increased in NK2R-deficient mice compared with that in wild-type mice, and the antitumor effects of poly I:C were abolished by NK2R absence. These findings suggest that IFN-γ-STAT1-mediated NK2R expression is involved in the induction of antitumor effector T cells in the tumor microenvironment, which contributes to the suppression of cancer cell tumorigenesis in vivo. In this study, we revealed that IFN-γ-STAT1-mediated NK2R expression is involved in the induction of antitumor effector CD8+ T cells in the tumor microenvironment, which contributes to suppressing the tumorigenesis of liver cancer cells in vivo.

[Lys5,MeLeu9,Nle10]-NKA(4-10) induces neurokinin 2 receptor mediated urination and defecation and neurokinin 1 receptor mediated flushing in rats: measured using the rapid detection voiding assay.

OBJECTIVES: Neurokinin 2 receptor (NK2R) agonists may be useful for treating bladder and bowel dysfunction via direct contraction of detrusor and gastrointestinal smooth muscle. The NK2R agonist [Lys5, MeLeu9, Nle10]-NKA(4-10) (LMN-NKA) induces urination and defecation, but also produces the potential side effect of dermal flushing in rats. Although LMN-NKA is a NK2R agonist, it also has affinity for neurokinin 1 receptors (NK1R). Therefore, the goal of this study was to determine the neurokinin receptor (NKR) subtypes responsible for LMN-NKA-induced urination, defecation, and flushing by blocking either NK2Rs or NK1Rs before LMN-NKA administration. METHODS: To accomplish this goal, we developed a simple high-throughput 'rapid detection voiding assay' to detect rapid-onset drug-induced urination and defecation in rats. In LMN-NKA dose-response experiments, LMN-NKA (10-100 µg/kg, subcutaneous) was injected and urination, defecation, and flushing were monitored for 30 min. For NKR antagonist experiments, vehicle, the NK2R antagonist GR159897, or the NK1R antagonist CP-99,994 were injected before an acclimation period. Following acclimation, saline or 100 µg/kg LMN-NKA were injected, and behavior was observed for 30 min. RESULTS: LMN-NKA produced dose-related increases in urination, defecation, and flushing. Blocking NK2Rs reduced urination and blocked defecation, without affecting flushing. Blocking NK1Rs did not change LMN-NKA-induced urination or defecation but reduced LMN-NKA-induced flushing. CONCLUSIONS: Using the rapid detection voiding assay we show that LMN-NKA-induced urination and defecation are mediated by NK2Rs, while flushing is mediated by NK1Rs. Therefore, drugs that are more selective for NK2 vs. NK1Rs should produce rapid-onset urination and defecation without producing the potential side effect of flushing.

Regional differences of tachykinin effects on smooth muscle and pacemaker potentials of the stomach, duodenum, ileum and colon of an emetic model, the house musk shrews.

BACKGROUND AND AIMS: The contractile effects of tachykinins on the gastrointestinal tract are well-known, but how they modulate slow-waves, particularly in species capable of emesis, remains largely unknown. We aimed to elucidate the effects of tachykinins on myoelectric and contractile activity of isolated gastrointestinal tissues of the Suncus murinus. METHODS: The effects of substance P (SP), neurokinin (NK)A, NKB and selective NK1 (CP122,721, CP99,994), NK2 (SR48,968, GR159,897) and NK3 (SB218,795, SB222,200) receptor antagonists on isolated stomach, duodenum, ileum and colon segments were studied. Mechanical contractile activity was recorded using isometric force displacement transducers. Electrical pacemaker activity was recorded using a microelectrode array. RESULTS: Compared with NKA, SP induced larger contractions in stomach tissue and smaller contractions in intestinal segments, where oscillation magnitudes increased in intestinal segments, but not the stomach. CP122,721 and GR159,897 inhibited electrical field stimulation-induced contractions of the stomach, ileum and colon. NKB and NK3 had minor effects on contractile activity. The inhibitory potencies of SP and NKA on the peristaltic frequency of the colon and ileum, respectively, were correlated with those on electrical pacemaker frequency. SP, NKA and NKB inhibited pacemaker activity of the duodenum and ileum, but increased that of the stomach and colon. SP elicited a dose-dependent contradictive pacemaker frequency response in the colon. CONCLUSION: This study revealed distinct effects of tachykinins on the mechanical and electrical properties of the stomach and colon vs. the proximal intestine, providing a unique aspect on neuromuscular correlation in terms of the effects of tachykinin on peristaltic and pacemaker activity in gastrointestinal-related symptoms.

Effects of Substance P and Neurokinin A on the Contractile Activity of Inflamed Porcine Uterus.

Disturbances in uterine contractile activity contribute to the development of inflammation, and recent evidence indicates that tachykinins, including substance P (SP) and neurokinin A (NKA), are involved in controlling uterine function. Here, we determined the effect of Escherichia coli (E. coli)-induced inflammation on expression of protein receptor subtypes for substance P (NK1R) and neurokinin A (NK2R) in the pig myometrium as well as their role in contractility of inflamed uterus. The severe acute endometritis developed in the E. coli group and the expression of NK1R and NK2R proteins increased in the myometrium. Compared to the pre-administration period, SP (10-6 M) reduced the amplitude and frequency in the myometrium of the E. coli group and the amplitude was higher and the frequency was lower versus other groups. NKA reduced the amplitude and increased the frequency in endometrium/myometrium of the E. coli group. In this group, the amplitude was lower and the frequency was higher than in the CON and SAL groups. Our research showed that NK2R (10-6 M) antagonist application abolished the NKA inhibitory effect on uterine amplitude. The application of the NK1R (10-5 M) antagonist together with SP revealed that the inhibitory effect of SP on uterine contractility is achieved independently of the NKR1. Additionally, taking into account the fact that NKA shows an inhibitory effect with the use of NK2R on uterine amplitude suggests the possibility of therapeutic use of the antagonist as a drug increasing uterine contractility in inflammation.

Ionic signalling mechanisms involved in neurokinin-3 receptor-mediated augmentation of fear-potentiated startle response in the basolateral amygdala.

The tachykinin peptides include substance P (SP), neurokinin A and neurokinin B, which interact with three G-protein-coupled neurokinin receptors, NK1Rs, NK2Rs and NK3Rs, respectively. Whereas high densities of NK3Rs have been detected in the basolateral amygdala (BLA), the functions of NK3Rs in this brain region have not been determined. We found that activation of NK3Rs by application of the selective agonist, senktide, persistently excited BLA principal neurons. NK3R-elicited excitation of BLA neurons was mediated by activation of a non-selective cation channel and depression of the inwardly rectifying K+ (Kir) channels. With selective channel blockers and knockout mice, we further showed that NK3R activation excited BLA neurons by depressing the G protein-activated inwardly rectifying K+ (GIRK) channels and activating TRPC4 and TRPC5 channels. The effects of NK3Rs required the functions of phospholipase Cß (PLCß), but were independent of intracellular Ca2+ release and protein kinase C. PLCß-mediated depletion of phosphatidylinositol 4,5-bisphosphate was involved in NK3R-induced excitation of BLA neurons. Microinjection of senktide into the BLA of rats augmented fear-potentiated startle (FPS) and this effect was blocked by prior injection of the selective NK3R antagonist SB 218795, suggesting that activation of NK3Rs in the BLA increased FPS. We further showed that TRPC4/5 and GIRK channels were involved in NK3R-elicited facilitation of FPS. Our results provide a cellular and molecular mechanism whereby NK3R activation excites BLA neurons and enhances FPS. KEY POINTS: Activation of NK3 receptors (NK3Rs) facilitates the excitability of principal neurons in rat basolateral amygdala (BLA). NK3R-induced excitation is mediated by inhibition of GIRK channels and activation of TRPC4/5 channels. Phospholipase Cß and depletion of phosphatidylinositol 4,5-bisphosphate are necessary for NK3R-mediated excitation of BLA principal neurons. Activation of NK3Rs in the BLA facilitates fear-potentiated startle response. GIRK channels and TRPC4/5 channels are involved in NK3R-mediated augmentation of fear-potentiated startle.

Neurokinin 1 and 2 receptors are involved in PGE2- and citric acid-induced cough and ventilatory responses.

Exposure to aerosolized citric acid (CA, 150 mM) and prostaglandin E2 (PGE2, 0.43 mM) for 10 min in guinea pigs reportedly produces the distinct cough patterns (Type I vs. II) and ventilatory responses (long-lasting hyperventilation vs. brief tachypnea) even though triggering the same cough numbers. Type I and II coughs are primarily mediated by activation of TRPV1 and EP3 receptors (a PGE2 receptor) of vagal C-fibers respectively. Substance P (SP) and neurokinin A (NKA) released by vagal pulmonary sensory fibers peripherally are capable of affecting CA-induced cough and ventilation via preferentially activating neurokinin 1 and 2 receptors (NK1R and NK2R) respectively. This study aimed to define the impacts of CA- and PGE2-exposure on pulmonary SP and NKA levels and the roles of NK1R and NK2R in modulating CA- and PGE2-evoked cough and ventilatory responses. In unanesthetized guinea pigs, we determined: (1) pulmonary SP and NKA contents induced by the CA- or PGE2-exposure; (2) effects of CP-99994 and SR-48968 (a NK1R and a NK2R antagonist respectively) given by intraperitoneal injection (IP) or aerosol inhalation (IH) on the CA- and PGE2-evoked cough and ventilatory responses; and (3) immunocytochemical expressions of NK1R/NK2R in vagal C-neurons labeled by TRPV1 or EP3 receptors. We found that CA- and PGE2-exposure evoked Type I and II cough respectively associated with different degrees of increases in pulmonary SP and NKA. Applications of CP-99994 and SR-48968 via IP and IH efficiently suppressed the cough responses to CA with less impact on the cough response to PGE2. These antagonists inhibited or blocked the ventilatory response to CA and caused hypoventilation in response to PGE2. Moreover, NK1R and NK2R were always co-expressed in vagal C-neurons labeled by TRPV1 or EP3 receptors. These results suggest that SP and NKA endogenously released by CA- and PGE2-exposure play important roles in generating the cough and ventilatory responses to CA and PGE2, at least in part, via activation of NK1R and NK2R expressed in vagal C-neurons (pulmonary C-neurons).

IFN-α/ß-mediated NK2R expression is related to the malignancy of colon cancer cells.

Neurokinin 2 receptor (NK2R), a G protein-coupled receptor for neurokinin A (NKA), a tachykinin family member, regulates various physiological functions including pain response, relaxation of smooth muscle, dilation of blood vessels, and vascular permeability. However, the precise role and regulation of NK2R expression in cancer cells have not been fully elucidated. In this study, we found that high NK2R gene expression was correlated with the poor survival of colorectal cancer patients, and Interferon (IFN-α/ß) stimulation significantly enhanced NK2R gene expression level of colon cancer cells in a Janus kinas 1/2 (JAK 1/2)-dependent manner. NKA stimulation augmented viability/proliferation and phosphorylation of Extracellular-signal-regulated kinase 1/2 (ERK1/2) levels of IFN-α/ß-treated colon cancer cells and NK2R blockade by using a selective antagonist reduced the proliferation in vitro. Administration of an NK2R antagonist alone or combined with polyinosinic-polycytidylic acid, a synthetic analog of double-stranded RNA, to CT26-bearing mice significantly suppressed tumorigenesis. NK2R-overexpressing CT26 cells showed enhanced tumorigenesis and metastatic colonization in both lung and liver after the inoculation into mice. These findings indicate that IFN-α/ß-mediated NK2R expression is related to the malignancy of colon cancer cells, suggesting that NK2R blockade may be a promising strategy for colon cancers.

Inflammatory and contractile profile in LPS-challenged equine isolated bronchi: Evidence for IL-6 as a potential target against AHR in equine asthma.

BACKGROUND: Airway inflammation and airway hyperresponsiveness (AHR) are pivotal characteristics of equine asthma. Lipopolysaccharide (LPS) may have a central role in modulating airway inflammation and dysfunction. Therefore, the aim of this study was to match the inflammatory and contractile profile in LPS-challenged equine isolated bronchi to identify molecular targets potentially suitable to counteract AHR in asthmatic horses. METHODS: Equine isolated bronchi were incubated overnight with LPS (0.1-100 ng/ml). The contractile response to electrical field stimulation (EFS) and the levels of cytokines, chemokines, and neurokinin A (NKA) were quantified. The role of capsaicin sensitive-sensory nerves, neurokinin-2 (NK2) receptor, transient receptor potential vanilloid type 1 receptors (TRPV1), and epithelium were also investigated. RESULTS: LPS 1 ng/ml elicited AHR to EFS (+238.17 ± 25.20% P < 0.001 vs. control). LPS significantly (P < 0.05 vs. control) increased the levels of IL-4 (+36.08 ± 1.62%), IL-5 (+38.60 ± 3.58%), IL-6 (+33.79 ± 2.59%), IL-13 (+40.91 ± 1.93%), IL-1ß (+1650.16 ± 71.16%), IL-33 (+88.14 ± 8.93%), TGF-ß (22.29 ± 1.03%), TNF-α (+56.13 ± 4.61%), CXCL-8 (+98.49 ± 17.70%), EOTAXIN (+32.26 ± 2.27%), MCP-1 (+49.63 ± 4.59%), RANTES (+36.38 ± 2.24%), and NKA (+112.81 ± 6.42%). Capsaicin sensitive-sensory nerves, NK2 receptor, and TRPV1 were generally involved in the LPS-mediated inflammation. Epithelium removal modulated the release of IL-1ß, IL-33, and TGF-ß. Only the levels of IL-6 fitted with AHR to a wide range of EFS frequencies, an effect significantly (P < 0.05) inhibited by anti-IL-6 antibody; exogenous IL-6 induced significant (P < 0.05) AHR to EFS similar to that elicited by LPS. CONCLUSION: Targeting IL-6 with specific antibody may represent an effective strategy to treat equine asthma, especially in those animals suffering from severe forms of this disease.

Neuropeptide Y, calcitonin gene-related peptide, and neurokinin A in brain regions of HAB rats correlate with anxiety-like behaviours.

Anxiety disorders are pervasive psychiatric disorders causing great suffering. The high (HAB) and low (LAB) anxiety-related behaviour rats were selectively bred to investigate neurobiological correlates of anxiety. We compared the level of neuropeptides relevant for anxiety- and depression-related behaviours in selected brain regions of HAB and LAB rats. Increased anxiety and depression-like behaviours of male and female HAB rats in the elevated plus-maze and forced swim tests were accompanied by elevated levels of neuropeptide Y (NPY) in the prefrontal (PFC), frontal (FC) and cingulate cortex (CCx), the striatum, and periaqueductal grey (PAG). Moreover, HAB rats displayed sex-dependent, elevated levels of calcitonin gene-related peptide (CGRP) in PFC, FC, CCx, hippocampus, and PAG. Higher neurokinin A (NKA) levels were detected in CCx, striatum, and PAG in HAB males and in CCx and hypothalamus in HAB females. Increased neurotensin was detected in CCx and PAG in HAB males and in hypothalamus in HAB females. Elevated corticotropin-releasing hormone (CRH) levels appeared in female HAB hypothalamus. Significant correlations were found between anxiety-like behaviour and NPY, CGRP, NKA, and neurotensin, particularly with NPY in CCx and striatum, CGRP in FC and hippocampus, and NKA in entorhinal cortex. This is the first report of NPY, CGRP, NKA, Neurotensin, and CRH measurements in brain regions of HAB and LAB rats, which showed widespread NPY and CGRP alterations in cortical regions, with NKA and neurotensin changes localised in sub-cortical areas. The results may contribute to elucidate pathophysiological mechanisms underlying anxiety and depression and should facilitate identifying novel therapeutic targets.

Neurokinins and their receptors in the rat trigeminal system: Differential localization and release with implications for migraine pain.

Substance P (SP) and calcitonin gene-related peptide (CGRP) have both been considered potential drug candidates in migraine therapy. In recent years, CGRP receptor inhibition has been established as an effective treatment, in particular as a prophylactic for chronic migraine. Curiously, inhibition of neurokinin receptor 1 (NK1R) failed to alleviate acute migraine attacks in clinical trials, and the neurokinins were consequently abandoned as potential antimigraine candidates. The reason behind this has remained enigmatic.Utilizing immunohistochemistry and semi-quantitative cell counts the expression of neurokinins and their associated receptors was examined in the rat trigeminal ganglion.Immunohistochemistry results revealed SP co-localization in CGRP positive neurons and C-fibres, where it mainly concentrated at boutons. Neurokinin A (NKA) was observed in a population of C-fibres and small neurons where it could co-localize with SP. In contrast, neurokinin B (NKB) did not co-localize with SP and was observed in large/medium sized neurons and Aδ-fibres. All neurokinin receptors (NK1-3R) were found to be expressed in a majority of trigeminal ganglion neurons and A-fibres.The functional release of SP and CGRP in the trigeminovascular system was stimulated with either 60 mM K+ or 100 nM capsaicin and measured with an enzyme-linked immunosorbent assay (ELISA). ELISA results established that SP can be released locally from trigeminovascular system. The released SP was comparatively minor compared to the CGRP release from stimulated dura mater, trigeminal ganglion neurons and fibres. We hypothesize that SP and CGRP signalling pathways may work in tandem to exacerbate painful stimuli in the TGV system.

Tachykinin NK2 antagonist for treatments of various disease states.

Tachykinin NK2 receptors are distributed in periphery, in the smooth muscle of the respiratory, gastrointestinal, genitourinary tract, and within the brain. Substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) are endogenous ligands for NK2 receptors and are active in the peripheral and central nervous systems. NK2 antagonists have the potential to reduce airway motor responses and prevent hyperactivity by inhibiting NKA-induced bronchoconstriction in asthmatic patients. Due to its abundance, peripherally and centrally, tachykinin NK2 receptor antagonists have high potential in treating various disease states ranging from asthma to irritable bowel syndrome, to detrusor hyperactivity, to anxiety. This review is an evaluation of NK2 receptor antagonists as possible therapeutics for a myriad of pharmacological treatments.

TRPM3 channel activation inhibits contraction of the isolated human ureter via CGRP released from sensory nerves.

AIMS: Sensory nerve activation modulates ureteral contractility by releasing neuropeptides including CGRP and neurokinin A (NKA). TRPM3 is a recently discovered thermosensitive channel expressed in nociceptive sensory neurons, and plays a key role in heat nociception and chronic pain. The aim of this study is to examine the role of TRPM3 activation in human ureter motility. MAIN METHOD: Human proximal ureters were obtained from fourteen patients undergoing nephrectomy. Spontaneous or NKA-evoked contractions of longitudinal ureter strips were recorded in an organ bath. Ureteral TRPM3 expression was examined by immunofluorescence. KEY FINDINGS: Spontaneous contractions were observed in 60% of examined strips. TRPM3 activation using pregnenolone sulphate (PS) or CIM0216 (specific TRPM3 agonists) dose-dependently reduced the frequency of spontaneous and NKA-evoked contractions, with IC50s of 241.7 µM and 4.4 µM, respectively. The inhibitory actions of TRPM3 agonists were mimicked by CGRP (10 to 100 nM) or a cAMP analogue (8-Br-cAMP; 1 mM). The inhibitory actions of TRPM3 agonists (300 µM PS or 30 µM CIM0216) were blocked by pretreatment with primidone (TRPM3 antagonist; 30 µM), tetrodotoxin (sodium channel blocker; 1 µM), olcegepant (CGRP receptor antagonist; 10 µM), or H89 (non-specific PKA inhibitor; 30 µM). TRPM3 was co-expressed with CGRP in nerves in the sub-urothelial and intermuscular regions of the ureter. SIGNIFICANCE: TRPM3 channels expressed on sensory terminals of the human ureter involve in inhibitory sensory neurotransmission and modulate ureter motility via the CGRP-cAMP-PKA signal pathway. Targeting TRPM3 may be a pharmacological strategy for promoting the ureter stone passage.

Gender-Related Differences of Tachykinin NK2 Receptor Expression and Activity in Human Colonic Smooth Muscle.

The tachykinin NK2 receptor plays a key role in gastrointestinal motor function. Enteric neurons release neurokinin A (NKA), which activates NK2 receptors on gastrointestinal smooth muscle, leading to contraction and increased motility. In patients with diarrhea-predominant irritable bowel syndrome, the NK2 receptor antagonist ibodutant had a greater therapeutic effect in females than males. The present study aimed to determine whether gender influences the expression and activity of NK2 receptors in human colonic smooth muscle. In vitro functional studies were performed to examine the contractile responses of colonic muscle strips to NKA and the selective NK2 receptor agonist [Lys5,MeLeu9,Nle10]NKA(4-10). Contractions were also measured in the presence of ibodutant to determine its antagonistic potency. The signal transduction pathways coupled to NK2 receptor activation were investigated using second messenger inhibitors. Western blot and fluorescent immunohistochemistry were conducted to determine the protein expression and localization of NK2 receptors. NK2 receptor-mediated contractility was greater in females compared with males. When against NKA, ibodutant was more potent in females. NK2 receptor expression increased with age in females, but not in males. Phospholipase C-mediated signaling was less prominent in females compared with males, whereas Ca2+ sensitization via Rho kinase and protein kinase C appeared to be the dominant pathway in both genders. The distribution of NK2 receptors in the human colon did not differ between the genders. Overall, gender differences exist in the expression and activity of NK2 receptors in colonic smooth muscle. These gender distinctions should be considered in the therapeutic development of NK2 receptor agents. SIGNIFICANCE STATEMENT: The tachykinin NK2 receptor has been identified as a therapeutic target for the treatment of bowel and bladder dysfunctions. The present study has revealed gender-related variations in NK2 receptor activity, signaling transduction pathways, antagonist potency, and changes in expression with age. These factors may underlie the gender differences in the treatment of diarrhea-predominant irritable bowel syndrome with NK2 receptor antagonists. Our findings highlight that gender differences should be considered in the therapeutic development of NK2 receptor agents.

Comparison of Neurokinin A, Substance P, Interleukin 8, and Matrix Metalloproteinase-8 Changes in Pulp tissue and Gingival Crevicular Fluid Samples of Healthy and Symptomatic Irreversible Pulpitis Teeth.

INTRODUTION: The aim of this study was to compare levels of neurokinin A (NKA), substance P (SP), interleukin (IL)-8, and matrix metalloproteinase-8 (MMP-8) in pulp tissue and gingival crevicular fluid (GCF) samples of healthy and symptomatic irreversible pulpitis teeth. METHODS: Forty patients diagnosed with healthy and symptomatic irreversible pulpitis teeth were included in this study. NKA, SP, IL-8, and MMP-8 levels were measured using the enzyme-linked immunosorbent assay test after pulp and GCF samples were obtained from healthy (n = 20) and symptomatic irreversible pulpitis teeth (n = 20). GCF sampling of 40 teeth was repeated 1 week later. Routine root canal treatment procedures of the teeth were performed, and the treatment process was completed. As a control group, GCF samples were taken from the contralateral teeth in both groups. Statistical analysis was performed using dependent and independent t tests, analysis of variance, Kruskal-Wallis, Mann-Whitney U tests, and Pearson correlation analysis. RESULTS: Comparing the groups, all mediator levels were significantly higher in the pulp samples in the pulpitis group compared with the healthy group (NKA: P < .001, SP: P = .005, IL-8: P < .001, and MMP-8: P < .001). Likewise, in the pulpitis group, all mediator levels were significantly higher in the first GCF samples compared with the healthy group (NKA: P = .01, SP: P < .001, IL-8: P = .001, and MMP-8: P < .001). CONCLUSIONS: It was observed that NKA, SP, IL-8, and MMP-8 increased significantly in pulp tissue and GCF specimens of symptomatic irreversible pulpitis teeth compared with pulp tissue and GCF specimens of healthy teeth. Second, it was determined that NKA, SP, IL-8, and MMP-8 levels decreased significantly in GCF samples in teeth diagnosed with symptomatic irreversible pulpitis 1 week after the removal of inflamed pulp. Finally, SP, IL-8, and MMP-8 levels were found to be higher in pulp tissue samples of the patients with symptomatic irreversible pulpitis with higher pain scores than those with low pain scores.

Effects of inhalation of sevoflurane at different concentrations on TRPV1 in airways of rats at different developmental stages.

Aim Determine changes in the expressions of the ion channel-TRPV1-and neuropeptides-NKA, NKB, calcitonin gene-related peptide (CGRP), and SP-in 14-, 21-, and 42-day-old rats after inhaling 1.5% and 2.6% sevoflurane. MAIN METHODS: A small in-house inhalation anesthesia chamber was designed to allow 14-, 21-, and 42-day-old rats inhale 1.5% and 2.6% sevoflurane, and rats in the control group inhaled carrier gas(1 L/min air +1 L/min O2). In addition, 14- and 21-day-old rats were pretreated with capsazepine, followed by inhalation of 1.5% and 2.6% sevoflurane or the carrier gas. The expression of TRPV1 in lung tissues was detected by Western blotting, whereas the expressions of NKA, NKB, CGRP, and SP in the trachea were detected by immunohistochemistry. KEY FINDINGS: After inhalation of 1.5% sevoflurane, the expression of TRPV1 in the lung tissues of 14- and 21-day-old rats was significantly increased compared with that in the control group, which was antagonized by capsazepine pretreatment. Moreover, inhalation of 1.5% sevoflurane markedly increased the expressions of NKA, NKB, CGRP, and SP in the trachea of 21-day-old rats and of NKB, CGRP, and SP in the trachea of 14-day-old rats. The expressions of these molecules were antagonized by capsazepine pretreatment. Conversely, inhalation of 2.6% sevoflurane decreased the expressions of NKA and NKB in the trachea of 42-day-old rats. SIGNIFICANCE: Sevoflurane did not upregulate the expression of TRPV1 in the airways of late-developing rats. This anesthetic may have a two-way effect on airways, resulting in considerable effects in pediatric clinical anesthesia management.

The role of neurokinin A and its receptor in the regulation of prolactin secretion by the anterior pituitary of cyclic pigs.

In pigs, plasma prolactin concentration markedly changes during the oestrous cycle and the regulation of its secretion is very complex. The contribution of neurokinins in this process has not been sufficiently delineated. The aim of the study was to examine the effects of neurokinin A (NKA) on prolactin synthesis and secretion in cyclic gilts. The expression of NKA precursor (Ppta) and receptor (Tacr2) genes as well as NKA and TACR proteins content in the porcine pituitaries (days 2-3, 9-10, 12-13, 15-16 and 19-20 of the cycle) was determined. Furthermore, the in vitro influence of NKA on the expression of prolactin (Prl), dopamine receptor (D2r), TRH receptor (Trhr) genes and prolactin secretion by the porcine pituitary cells (days 9-10, 15-16 and 19-20 of the cycle) was assessed. The expression of Ppta and Tacr2 as well as NKA and TACR proteins in the pituitary tissue has been changing throughout the oestrous cycle. NKA affected in vitro the expression of studied genes and prolactin secretion depending on the stage of the cycle, dose of NKA and/or duration of the cell incubation. Altogether, the study indicates that NKA is engaged in the modulation of prolactin secretion in the pig during the oestrous cycle.