Role of NK1 and NK2 receptors in mouse gastric mechanical activity.
ABSTRACT: 1. The aim of the present study was to examine the role of NK1 and NK2 receptors in the control of mechanical activity of mouse stomach. In this view, the motor effects induced by NK1 and NK2 receptor agonists and antagonists were analyzed, measuring motility as intraluminal pressure changes in mouse-isolated stomach preparations. In parallel, immunohistochemical studies were performed to identify the location of NK1 and NK2 receptors on myenteric neurons and smooth muscle cells. 2. Substance P (SP) induced biphasic effects: a contraction followed by relaxation; neurokinin A (NKA) and [beta-Ala8]-NKA(4-10), selective agonist of NK2 receptors, evoked concentration-dependent contractions, whereas [Sar9, Met(O2)11]-SP, selective agonist of NK1 receptors, induced concentration-dependent relaxation. 3. SR48968, NK2 receptor antagonist, did not modify the spontaneous activity and reduced the contractile effects induced by tachykinins without affecting the relaxation. SR140333, NK1 receptor antagonist, did not modify the spontaneous activity and antagonized the relaxant response to tachykinins, failing to affect the contractile effects. 4. The relaxation to SP or to [Sar9, Met(O2)11]-SP was abolished by tetrodotoxin (TTX) and significantly reduced by N(omega)-nitro-L-arginine methyl ester (L-NAME). 5. NK2-immunoreactivity (NK2-IR) was seen at the level of the smooth muscle cells of both circular and longitudinal muscle layers. NK1-immunoreactive (NK1-IR) neurons were seen in the myenteric ganglia and NK1/nNOS double labeling revealed that some neurons were both NK1-IR and nNOS-IR. 6. These results suggest that, in mouse stomach, NK1 receptors, causing relaxant responses, are present on nitrergic inhibitory myenteric neurons, whereas NK2 receptors, mediating contractile responses, are present at muscular level.
Project description:Gastric ulceration, a focal tissue damage accompanied by inflammation, can influence other parts of the stomach. Substance P and its receptors are strongly involved in regulation of gastrointestinal motility, secretion and inflammation. The enteric nervous system is one of the regulators of gastrointestinal functioning and contributes to tissue response to the pathology. The pig, an omnivorous animal, is a valuable species for gastrointestinal experiments. Thus, the objective of the study was to verify whether the antral ulceration induces changes in the expression of substance P and tachykinin receptors in the neighboring (antrum) and distanced (corpus, pylorus) porcine gastric tissues and therein localized myenteric and submucosal perikarya as well as in the intrinsic descending neurons supplying pyloric sphincter. The experiment was performed on healthy pigs and pigs with experimentally induced gastric ulcers. Stomach samples from the corpus, antrum (adjacent to the ulcer in experimental pigs) and pylorus were analyzed by: (1) double immunofluorescence for changes in the number of SP-positive myenteric and submucosal neurons (2) Real-Time PCR for changes in expression of mRNA encoding SP and Nk1, Nk2, Nk3 receptors. Additionally, gastric descending neurons supplying pyloric sphincter were immunostained for SP. In experimental animals, only the number of SP-positive myenteric perikarya significantly increased in all stomach localizations studied. Q-PCR revealed increased expression for: SP, Nk1, Nk3 in the corpus; Nk2 and Nk3 in the pylorus; In the antrum, expression of Nk3 was increased but Nk2-decreased. Antral ulcers induced significant changes in the expression of SP and tachykinin receptors in the wide stomach area indicating sophisticated tissue reaction.
Project description:A tachykinin peptide was isolated from an extract of the intestine of the European green frog, Rana ridibunda, and its primary structure was established as: His-Lys-Leu-Asp-Ser-Phe-Ile-Gly-Leu-Met.CONH2. This sequence was confirmed by chemical synthesis and shows two amino acid substitutions (leucine for threonine at position 3 and isoleucine for valine at position 7) compared with neurokinin A. Binding parameters for synthetic [Leu3,Ile7]neurokinin A and mammalian tachykinins were compared using receptor-selective radioligands and crude membranes from tissues enriched in the NK1, NK2 and NK3 receptors. [Leu3,Ile7]Neurokinin A was approx. 3-fold less potent than substance P in inhibiting the binding of 125I-labelled [Sar9,Met(O2)11]substance P (labelled with Bolton-Hunter reagent) to rat submandibular gland (NK1 receptor), 8-fold less potent than neurokinin A in inhibiting the binding of [2-[125I]iodohistidine1]neurokinin A to rat stomach fundus (NK2 receptor) and 6-fold less potent than neurokinin B in inhibiting the binding of 125I-Bolton-Hunter-labelled scyliorhinin II to rat brain (NK3 receptor). Thus the frog neurokinin A-related peptide shows moderate affinity but lack of selectivity for all three tachykinin-binding sites in rat tissues. This non-selectivity is similar to that displayed by the molluscan tachykinin, eledoisin, which also contains an isoleucine residue in the corresponding position in the molecule.
Project description:There is growing evidence for a pivotal role for tachykinins in gut neuroimmune interactions.To determine whether NK1, NK2, and NK3 tachykinin receptors are involved in milk protein induced allergic sensitisation.Eight groups of 12 Dunkin-Hartley guinea pigs (250-300 g) were used. Four groups were sensitised to milk proteins for three weeks. During this period, these animals were injected intraperitoneally each day with NK1 (SR 140333; 0.3 mg/kg), NK2 (SR 48968; 5 mg/kg), or NK3 (SR 142801; 5 mg/kg) receptor antagonist or vehicle. The fifth group had water available instead of milk and was used as a non-sensitised control. The three other groups received the NK receptor antagonists for three weeks but were not sensitised to milk proteins.Sensitised animals treated with NK1 and NK3 receptor antagonists had both lower IgE and IgG serum titres, evaluated by passive cutaneous anaphylaxis, and lower specific IgG serum titres, determined by enzyme linked immunosorbent assay (ELISA), than vehicle treated animals. Sensitisation induced an increase in intestinal mast cell number which was abolished by treatment with the NK1 receptor antagonist. Antigenic challenge-induced jejunal hypersecretion was also blocked by treatment with the NK1 receptor antagonist.In guinea pigs, NK1 and NK3 but not NK2 receptors are involved in sensitisation to cow's milk. However, NK1 but not NK3 receptor antagonists abolish both the hypermastocytosis induced by food allergy and the hypersecretion induced by antigenic challenge, suggesting different roles for NK1 and NK3 receptors in the mechanisms of sensitisation to beta-lactoglobulin.
Project description:BACKGROUND AND PURPOSE: Peptide welding technology (PWT) is a novel chemical strategy that allows the synthesis of multibranched peptides with high yield, purity and reproducibility. Using this technique, we have synthesized and pharmacologically characterized the tetrabranched derivatives of the tachykinins, substance P (SP), neurokinin A (NKA) and B (NKB). EXPERIMENTAL APPROACH: The following in vitro assays were used: calcium mobilization in cells expressing human recombinant NK receptors, BRET studies of G-protein - NK1 receptor interaction, guinea pig ileum and rat urinary bladder bioassays. Nociceptive behavioural response experiments were performed in mice following intrathecal injection of PWT2-SP. KEY RESULTS: In calcium mobilization studies, PWT tachykinin derivatives behaved as full agonists at NK receptors with a selectivity profile similar to that of the natural peptides. NK receptor antagonists display similar potency values when tested against PWT2 derivatives and natural peptides. In BRET and bioassay experiments PWT2-SP mimicked the effects of SP with similar potency, maximal effects and sensitivity to aprepitant. After intrathecal administration in mice, PWT2-SP mimicked the nociceptive effects of SP, but with higher potency and a longer-lasting action. Aprepitant counteracted the effects of PWT2-SP?in vivo. CONCLUSIONS AND IMPLICATIONS: The present study has shown that the PWT technology can be successfully applied to the peptide sequence of tachykinins to generate tetrabranched derivatives characterized with a pharmacological profile similar to the native peptides. In vivo, PWT2-SP displayed higher potency and a marked prolongation of action, compared with SP.
Project description:Hepatocyte growth factor (HGF) is an activating ligand of the Met receptor tyrosine kinase, whose activity is essential for normal tissue development and organ regeneration but abnormal activation of Met has been implicated in growth, invasion, and metastasis of many types of solid tumors. HGF has two natural splice variants, NK1 and NK2, which contain the N-terminal domain (N) and the first kringle (K1) or the first two kringle domains of HGF. NK1, which is a Met agonist, forms a head-to-tail dimer complex in crystal structures and mutations in the NK1 dimer interface convert NK1 to a Met antagonist. In contrast, NK2 is a Met antagonist, capable of inhibiting HGF's activity in cell proliferation without clear mechanism. Here we report the crystal structure of NK2, which forms a "closed" monomeric conformation through interdomain interactions between the N- domain and the second kringle domain (K2). Mutations that were designed to open up the NK2 closed conformation by disrupting the N/K2 interface convert NK2 from a Met antagonist to an agonist. Remarkably, this mutated NK2 agonist can be converted back to an antagonist by a mutation that disrupts the NK1/NK1 dimer interface. These results reveal the molecular determinants that regulate the agonist/antagonist properties of HGF NK2 and provide critical insights into the dimerization mechanism that regulates the Met receptor activation by HGF.
Project description:NK1 and NK3 receptors do not appear to play significant roles in normal GI functions, but both may be involved in defensive or pathological processes. NK1 receptor antagonists are antiemetic, operating via vagal sensory and motor systems, so there is a need to study their effects on other gastro-vagal functions thought to play roles in functional bowel disorders. Interactions between NK1 receptors and enteric nonadrenergic, noncholinergic motorneurones suggest a need to explore the role of this receptor in disrupted colonic motility. NK1 receptor antagonism does not exert consistent analgesic activity in humans, but similar studies have not been carried out against pain of GI origin, where NK1 receptors may have additional influences on mucosal inflammatory or "irritant" processes. NK3 receptors mediate certain disruptions of intestinal motility. The activity may be driven by tachykinins released from intrinsic primary afferent neurones (IPANs), which induce slow EPSP activity in connecting IPANs and hence, a degree of hypersensitivity within the enteric nervous system. The same process is also proposed to increase C-fibre sensitivity, either indirectly or directly. Thus, NK3 receptor antagonists inhibit intestinal nociception via a "peripheral" mechanism that may be intestine-specific. Studies with talnetant and other selective NK3 receptor antagonists are, therefore, revealing an exciting and novel pathway by which pathological changes in intestinal motility and nociception can be induced, suggesting a role for NK3 receptor antagonism in irritable bowel syndrome.
Project description:Opioid agonists are well-established analgesics, widely prescribed for acute but also chronic pain. However, their efficiency comes with the price of drastically impacting side effects that are inherently linked to their prolonged use. To answer these liabilities, designed multiple ligands (DMLs) offer a promising strategy by co-targeting opioid and non-opioid signaling pathways involved in nociception. Despite being intimately linked to the Substance P (SP)/neurokinin 1 (NK1) system, which is broadly examined for pain treatment, the neurokinin receptors NK2 and NK3 have so far been neglected in such DMLs. Herein, a series of newly designed opioid agonist-NK2 or -NK3 antagonists is reported. A selection of reported peptidic, pseudo-peptidic, and non-peptide neurokinin NK2 and NK3 ligands were covalently linked to the peptidic μ-opioid selective pharmacophore Dmt-DALDA (H-Dmt-d-Arg-Phe-Lys-NH<sub>2</sub>) and the dual μ/δ opioid agonist H-Dmt-d-Arg-Aba-βAla-NH<sub>2</sub> (KGOP01). Opioid binding assays unequivocally demonstrated that only hybrids <b>SBL-OPNK-5</b>, <b>SBL-OPNK-7</b> and <b>SBL-OPNK-9</b>, bearing the KGOP01 scaffold, conserved nanomolar range μ-opioid receptor (MOR) affinity, and slightly reduced affinity for the δ-opioid receptor (DOR). Moreover, NK binding experiments proved that compounds <b>SBL-OPNK-5</b>, <b>SBL-OPNK-7</b>, and <b>SBL-OPNK-9</b> exhibited (sub)nanomolar binding affinity for NK2 and NK3, opening promising opportunities for the design of next-generation opioid hybrids.
Project description:Breast cancer remains the cancer with the highest mortality among women in the United States. Peptides derived from the oncogenic Tac1 gene (full transcript: betaPPT-A) stimulate the proliferation of breast cancer cells (BCCs) via seven-transmembrane G protein-coupled neurokinin 1 (NK1) and NK2 receptors. The NK1 gene could generate full-length (NK1-FL) and truncated (NK1-Tr) transcripts. NK1-Tr lacks 100 residues in their cytoplasmic end, could couple to G proteins, and shows reduced efficiency with respect to internalization and desensitization. This study reports on a role of NK1-Tr in the transformation of nontumorigenic breast cells, and investigates whether Tac1 expression is linked to the generation of NK1-Tr. Western blots and Northern analyses showed coexpressions of NK1-Tr and NK1-FL in BCCs (cell lines and primary cells from patients with different stages of breast cancer). Stable transfections of betaPPT-A or NK1-Tr expression vectors in nontumorigenic cells showed each induces the expression of the other, consequently resulting in a transformed phenotype. Analyses with microarrays indicate similar patterns of cytokine production by NK1-Tr transfectants and BCCs, but not NK1-FL transfectants. These observations indicate tumor-promoting properties by NK1-Tr, but not NK1-FL. Overall, the oncogenic property of Tac1 in breast cells involves concomitant expression of NK1-Tr and vice versa, consequently leading to the production of cytokines with growth promoting functions.
Project description:Tachykinins (TKs) are a family of peptides involved in the peripheral and central regulation of urinary functions through the stimulation of neurokinin (NK) NK1, NK2 and NK3 receptors. Recent evidence indicates that NK1 receptors are required in antigen-induced cystitis. Therefore, determining the regulatory network downstream NK1 receptor activation is a key step toward understanding the role of TKs in inflammation. For this purpose, we used a Transcriptional Regulatory Network Analysis (TRNA) to identify biologically relevant transcriptional regulatory elements (TRE) that underline the NK1-dependent gene expression in bladder responses to inflammation. Gene expression analysis was obtained using the urinary bladder isolated from WT and NK1-R-/- mice that were stimulated with intravesical instillation of saline or antigen challenge (in sensitized mice) in order to promote cystitis. Based on cDNA array results, we selected a cluster of genes that was dependent on NK1 receptors for their up-regulation in response to inflammation. Next, we used PAINT database to retrieve upstream promoter sequences for those NK1-R-dependent genes and to identify TREs on those promoters. Finally, TREs were enriched statistically by selecting only those that were significantly expressed and a regulatory network downstream of NK1 receptor activation was determined. This work indicates an overriding participation of NK1 receptors in bladder inflammation, provides a working model for the involvement of transcription regulators such as AP1, NF-kB, and Nkx-2.5, and evokes testable hypotheses regarding the regulatory network downstream of NK1 receptor activation. Keywords: URINARY BLADDER INFLAMMATION Overall design: All mice in this study were sensitized with 1 µg DNP4-human serum albumin (HSA) in 1 mg alum on days 0, 7, 14, and 21, intraperitoneally (i.p.). In normal mice, this protocol induces sustained levels of IgE antibodies up to 56 days post-sensitization (20). One week after the last sensitization, cystitis was induced. Briefly, sensitized WT and NK1R-/- mice were anesthetized (ketamine 40 mg/kg and xylazine 2.5 mg/kg, i.p.), then transurethrally catheterized (24 Ga.; 3/4 in; Angiocath, Becton Dickson, Sandy, Utah), and the urine was drained by applying slight digital pressure to the lower abdomen. The urinary bladders were instilled with 200 µl of pyrogen-free saline or DNP4-OVA (1 µg/ml). One, four, and twenty-four hours after instillation, mice were sacrificed with pentobarbital (100 mg/kg, i.p.) and bladders were removed rapidly. Gene expression was determined using Clontech 1.2 K arrays and genes specifically regulated in the WT were compared with those upregulated in NK1 KO. We have employed a bioinformatics approach to hypothesize functionally relevant transcriptional regulatory elements (TREs) of NK1R-dependent and -independent genes. We used the Promoter Analysis and Interaction Network Toolset (PAINT) available online at http://www.dbi.tju.edu/dbi/tools/paint (58). PAINT is a suite of bioinformatics and computational tools that integrate functional genomics information, as is the case of our microarray-based gene expression data, with genomic sequence and TRE data to derive hypotheses on the TREs relevant to the biological function under study. The TRE hypotheses are generated from statistical enrichment analysis and are defined as those TREs that are significantly enriched such as NK1R-dependent and –independent genes over random occurrence in the gene groups (58). The random occurrence is relative to a ‘reference’: all the genes on the microarray. Employing the microarray accounts for any ‘bias’ present in the genes on the microarray relative to entire genome and guards from incorrectly concluding that certain TREs are relevant to the current experiment.
Project description:Hepatocyte growth factor (HGF) is a pluripotent secreted protein that stimulates a wide array of cellular targets, including hepatocytes and other epithelial cells, melanocytes, endothelial and haematopoietic cells. Multiple mRNA species transcribed from a single HGF gene encode at least three distinct proteins: the full-length HGF protein and two truncated HGF isoforms that encompass the N-terminal (N) domain through kringle 1 (NK1) or through kringle 2 (NK2). We report the high-level expression in Escherichia coli of NK1 and NK2, as well as the individual kringle 1 (K1) and N domains of HGF. All proteins accumulated as insoluble aggregates that were solubilized, folded and purified in high yield using a simple procedure that included two gel-filtration steps. Characterization of the purified proteins indicated chemical and physical homogeneity, and analysis by CD suggested native conformations. Although the K1 and N-terminal domains of HGF have limited biological activity, spectroscopic evidence indicated that the conformation of each matched that observed when the domains were components of biologically active NK1. Both NK1 and NK2 produced in bacteria were functionally equivalent to proteins generated by eukaryotic systems, as indicated by mitogenicity, cell scatter, and receptor binding and activation assays. These data indicate that all four bacterially produced HGF derivatives are well suited for detailed structural analysis.