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Ore, Mikhail. "Neurokinin-1 receptor: neurokinin-1 receptor, purification and refolding." Doctoral thesis, Universitat Autònoma de Barcelona, 2012. http://hdl.handle.net/10803/96874.
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El receptor Neuroquinin-1 (NK1R) és un GPCR que es troba en el sistema nerviós central i perifèric dels vertebrats i és responsable dels processos fisiològics com la transmissió de dolor, secreció endocrina i exocrina, vasodilatació, modulació de la proliferació cel·lular i molts altres. Els antagonistes del NK1R poden ser potencials analgèsics i antidepressius i també poden ser utilitzats per al tractament del trastorn bipolar, l'alcoholisme, càncer, malalties del sistema immune i algunes infeccions. Les tècniques espectroscòpiques i les estructurals d'alta resolució com NMR i la cristal·lografia requereixen quantitats de l'ordre de mil·ligrams del receptor actiu purificat. Una de les estratègies que permet produir els GPCRs recombinants per estudis estructurals és el sistema d'expressió en E.coli. No obstant, molts GPCRs degut al seu efecte tòxic per a la cèl·lula bacteriana s'expressen en forma dels cossos d'inclusió i han de ser sotmesos al procés de renaturalització. La renaturalització dels GPCRs és una tasca complexa que implica complexos ajustaments dels tampons.
La primera part d'aquest treball s'ha centrat en la renaturalització de las formes truncades hNK1R-366 i hNK1R-311 del receptor expressades en cossos d'inclusió d'E.coli. Per a l'obtenció del receptor ben plegat hem establert un original protocol de la renaturalització en columna. Hem utilitzat diferents tècniques espectroscòpiques per a estudiar el receptor renaturalitzat. Els resultats de CD han demostrat que el hNK1R-366 i el hNK1R-311 renaturalitzats en DDM presenten un percentatge d'hèlix-α similar al de la rodopsina extreta de retines bovines i solubilitzada en DDM. En els estudis de fluorescència intrínseca de triptòfans a baixes concentracions del GuHCl hem pogut observar el desplaçament cap el blau en l'espectre d'emissió, típic de triptòfans que es troben en un entorn hidrofòbic. A més a més, els espectres d'emissió del hNK1R-366 expressat en cèl·lules COS-1 i solubilitzat en DDM presenten el màxim d'emissió a 335 nm, molt similar al del receptor renaturalitzat a partir dels cossos d'inclusió, indicant la seva correcte renaturalització. El hNK1R-366, renaturalitzat en tampó fisiològic presenta agregació en 24 hores. No obstant, la presència de 0.05% DDM és capaç d'estabilitzar el receptor. Els assajos de radioligand binding de saturació del hNK1R-366 renaturalitzat indiquen que el receptor actiu constitueix l’1% de la proteïna total de la mostra. No obstant, la unió de la SP al receptor en el rang de nanomols és significatiu i és un resultat important, donat que, per primera vegada s’ha obtingut NK1 funcional a partir de E.coli. Per altre costat, no hem pogut obtenir una corba de saturació del hNK1R-311, degut possiblement al plegament defectuós del receptor per falta dels darrers 96 residus.
La segona part d'aquest estudi està centrada en l'expressió, purificació i caracterització estructural del C-terminal del receptor hNK1. El domini C-terminal és important per l'acoblament de la proteïna G i la β-arrestina, i també és essencial per a la dessensibilització, internalització i reciclatge del receptor. No obstant, el paper d'aquest domini ha estat infravalorat pels investigadors durant molt temps i existeix poca informació sobre la seva estructura. Els estudis espectroscòpics de UV i de fluorescència posen de manifest anomalies en l'absorbància a 292 nm i en l'emissió intrínseca de les tirosines a 345 nm, atribuïdes a formes ionitzades de l’aminoàcid, degut a la seva proximitat a grups carboxil de residus glutàmics o aspàrtics. A partir de la predicció de l'estructura secundària i terciària i dels resultats dels estudis espectroscòpics hem proposat un model tridimensional pel C-terminal del hNK1 que conté: 25% d’hèlix-α, 27% d’estructura desordenada i 48% de fulles-β i girs-β. En conjunt, els resultat obtinguts, indiquen que el C-terminal del hNK1R no és un domini desordenat, sinó que té una estructura secundària i terciària clarament definides que poden relacionar amb les seves funcions.Neurokinin-1 receptor (NK1R) is a GPCR found in the central and peripheral nervous system of vertebrates, responsible for such physiological processes as pain transmission, exocrine and endocrine secretion, vasodilatation, modulation of cell proliferation and many others. NK1R antagonists could be potential analgesics and anti-depressants and may also be used for treatment of bipolar disorder, alcoholism, cancer, immune system diseases and selected infections. Spectroscopic studies and high resolution structural techniques, as NMR and crystallography, require milligram amounts of active purified receptor. One of the strategies to produce recombinant GPCRs for structural studies is an E.coli expression system. However, many GPCRs due to their toxic effect for bacterial host cell are expressed in form of inclusion bodies and require refolding. The refolding of GPCRs is a complicated task that requires screening and adjustment of buffer conditions. The first part of this work was centered on the refolding of hNK1R-366 and hNK1R-311 truncated forms expressed in E.coli inclusion bodies. To obtain properly folded receptor, we established an original on-column refolding protocol. Different spectroscopic techniques were applied to study the refolded receptor. The results obtained from CD measurements showed that hNK1R-366 refolded in DDM presents similar α-helical content as rhodopsin extracted from bovine retinas and solubilized in non-denaturing DDM micelles. In the intrinsic tryptophan fluorescence studies, at low concentrations of GuHCl we observed a blue-shift in the emission spectrum peak, typical for tryptophan in hydrophobic environment. Furthermore, the emission spectra of hNK1R-366 expressed in COS-1 cells and solubilized in DDM micelles show very similar emission maximum around 335 nm to that of the receptor refolded from the inclusion bodies, which may be indicative of proper protein refolding. The refolded in physiological buffer hNK1R-366 was prone to aggregate in about 24 hours, however, the presence of 0.05% DDM was found to stabilize the receptor. Saturation radioligand-binding assays for the refolded hNK1R-366 showed that the amount of the active receptor is about 1% of the total protein the sample. However, the binding of SP to the refolded hNK1R-366 in nanomolar range is significant and can be considered as a promising result, since until now the intents to produce any detectable amounts of functional NK1 receptor in E. coli were unsuccessful. On the other hand, we were unable to get any saturation binding curve for hNK1R-311 truncated form of the receptor, which could be explained by incorrect folding caused by the lack of 96 residues of the C-terminus of the receptor. The second part of the present study is centered on hNK1R C-terminus expression, purification and characterization to elucidate its structural properties. The C-terminus domain seems to be essential for the coupling to corresponding G protein and β-arrestin, and is essential for receptor desensitization, internalization and recycling. However, the role of this domain was underestimated by researchers for a long time and as a result very little information is known about its structure. UV and fluorescence spectroscopic studies revealed abnormal tyrosine red-shifted absorbance band at 292 nm and intrinsic tyrosine emission at 345 nm which could be attributed to ionized form of tyrosine and possibly arises from the proximity of one or more tyrosines to carboxyl groups of glutamic o aspartic residues. Based on secondary and tertiary structure prediction as well as on the results of spectroscopic studies we propose a 3D-model for hNK1R C-terminus. Th following assignment of the secondary structure was made: 25% α-helix, 27% unordered structure, 48% β-sheets and β-turns. The obtained ressults give evidence that hNK1R C-terminus is not an unordered region but has clearly defined secondary and tertiary structures which certainly are tightly related to its multiple functions.
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Carelse, Tofa Kashefa. "Molecular genetic analysis of the neurokinin B (TAC3) and neurokinin B receptor (TAC3) genes as candidates for pre-eclampsia." Thesis, Stellenbosch : Stellenbosch University, 2004. http://hdl.handle.net/10019.1/50029.
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Thesis (MSc)--Stellenbosch University, 2004.ENGLISH ABSTRACT: Hypertensive conditions of pregnancy, such as pre-eclampsia, are the principal direct cause of maternal morbidity and mortality and affect up to 10% of first pregnancies worldwide. The placenta is vital in the pathogenesis of pre-eclampsia since the condition only occurs in the presence of placental tissue and the only cure is delivery of the placenta and the fetus. It has been hypothesised that the placenta may be the source of a circulating factor(s), which transports freely in the maternal system, resulting in the multi-systemic and immunological responses that are characteristic of pre-eclampsia. Among the potential "circulating" candidates currently being investigated worldwide, is the tachykinin member, neurokinin B (NKB). The aim of this project was to use a novel approach and investigate the role of Neurokinin B in pre-eclampsia on a genetic level. This would be achieved by bioinformatie characterisation of the neurokinin B (TAC3) and neurokinin B receptor (TACR3) genes. Samples from thirty pre-eclampsia patients (of whom 10 also had abruptio placentae) and twenty control individuals were used for mutation detection analysis involving Multiphor gel electrophoresis and automated sequencing. Three sequence variants were identified in the TAC3 gene and include: (i) 5' UTR variant (-25 c-t); (ii) intronic variant IVS3-53 (t-g) and (iii) 3' UTR variant exon 7 (479, t-c). Only the -25 c-t variant had been reported before (SNP database). A further two variants were identified in the TACR3 gene: (i) exon 3 variant (nt 857, a-t) and (ii) 3' UTR variant, amplicon 5b (nt 1471, t-c), of which the latter had previously been reported in the SNP database. In the analysis of allele and genotype frequencies, only variant homozygosity for TAC3 -25 c-t could be associated with increased risk of pre-eclampsia (RR 3.33, p=0.03). Follow-up work will include extended genotyping in further stratified and larger patient cohorts and transfection studies to assess splicing potential and functional consequences of the mutant alleles. These data represent the first documented mutation screen of the TAC3 and TACR3 genes and report novel variants in patients with pre-eclampsia. This study contributes to the knowledge of neurokinin B as a circulatory molecule and confirms the heterogeneity of pre-eclampsia.
AFRIKAANSE OPSOMMING: Die belangrikste direkte oorsaak van moedersterftes is hipertensiewe toestande in swangerskap, insluitende pre-eklampsie. Hierdie toestande kompliseer wêreldwyd 10% van alle swangerskappe. Die plasenta is kardinaal in die ontwikkeling van die siekte aangesien dit slegs voorkom terwyl die plasenta in-situ is en die simptome opklaar na verlossing van die plasenta. 'n Moontlike hipotese is dat die plasenta 'n sirkulerende agens afskei wat in die moederlike sisteem beland en die uiteenlopende multi-sistemiese simptome en tekens van die siekte veroorsaak, asook aktivering van die immuunsisteem. Een van die moontlike kandidate wat tans wêreldwyd ondersoek word as moontlike sirkulerende agens, is Neurokinien B (NKB), 'n lid van die Tachikinien familie. Die unieke benadering van hierdie projek was om die rol van Neurokinien B in pre-eklampsie te ondersoek op 'n genetiese grondslag. Dit is bereik deur bio-informatiewe karakterisering van die neurokinien B (TAC3) en neurokinien B reseptor (TACR3) en deur mutasie sifting op DNA monsters van 30 pasiënte met pre-eklampsie (waarvan 10 ook abruptio placentae gehad het) en twintig kontrole individue met behulp van Multiphor gel elektroforese en ge-outomatiseerde volgorde bepaling. Drie volgorde variasies is geïdentifiseer in die TAC3 geen en sluit in: (i) 5' UTR variant (-25 c-t); (ii) introniese variant IVS3-53 (t-g) en (iii) 3' UTR variant in ekson 7 (479, t-e). Slegs die -25 c-t variasie is voorheen raporteer (SNP databasis). Nog twee variante is ook gevind in die TACR3 geen: (i) ekson 3 variant (nt 857, a-t) en (ii) 3' UTR variant, amplikon 5b (nt 1471, t-e); hierdie laaste een is al in die SNP databasis raporteer. In 'n analise van genotipe en allele frekwensies is slegs homosigositeit vir variant TAC3 -25 c-t geassosieër met 'n verhoogde risiko vir preeklampsie (RR 3.33, p=0.03). Verdere werk sal nou fokus op die genotipering van groter en gestratifiseerde pasiënt kohorte en transfeksie studies om splitsing potensiaal en funksionele gevolge van mutante allele te ondersoek. Hierdie data is die eerste gedokumenteerde mutasie sifting van die TAC3 en TACR3 gene en verslag word gelewer van unieke variasies in pasiënte met pre-eklampsie.
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Sandoval-Guzman, Tatiana. "Neurokinin B and the hypothalamic regulation of reproduction." Diss., The University of Arizona, 2003. http://hdl.handle.net/10150/280447.
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The morphology and gene expression of neurokinin B (NKB) neurons is altered in the human infundibular (arcuate) nucleus in association with the ovarian failure of menopause. Also, gonadotropin releasing-hormone (GnRH) mRNA is elevated and proopiomelanocortin (POMC) mRNA decreased. To determine if loss of ovarian steroids could produce comparable changes in gene expression in primates, we measured the effects of ovariectomy on NKB and GnRH in young cynomolgus monkeys. We also measured POMC gene expression, serum leptin and body weight to examine the consequences of ovariectomy on energy balance. Neurokinin B neurons in the infundibular nucleus of ovariectomized monkeys were larger, more numerous and displayed increased levels of NKB mRNA than the intact controls. Ovariectomy increased the number of neurons expressing GnRH gene transcripts. In contrast, the energy balance parameters were unchanged by ovariectomy. This study provides strong support for the hypothesis that ovarian failure contributes to the morphological changes and increased NKB and GnRH gene expression observed in postmenopausal women. We hypothesized that hypothalamic NKB neurons participate in the hypothalamic circuitry regulating LH. We determined if intracerebral infusion of a NK 3 receptor agonist alters serum LH in the ovariectomized estrogen-treated rat. A significant inhibition of serum LH was observed after senktide injection, accompanied by changes in Fos expression in medial preoptic area, arcuate, paraventricular and supraoptic nuclei. This study provides evidence that stimulation of the NK3 receptor may inhibit LH secretion via activation of hypothalamic neurons. To further investigate the role of NKB in gonadotropin regulation, we infused an antisense oligodeoxynucleotide targeted to the NKB gene in gonadectomized rats. In support of our hypothesis, the downregulation of NKB decreased serum LH by 25%. To analize the participation of the NKB receptor, NK3, we targeted an antisense to the receptor. Rats injected with the NK3 antisense exhibited no change in serum LH. Furthermore, injection of SB-222200, a NK3 antagonist, did not modify serum LH. These data suggest that NKB may regulate gonadotropin secretion through more than one receptor. Taken together, these studies provide some of the first detailed information on the relationship between NKB neurons, and the reproductive axis.
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Dacks, Penny Ann Frances. "THE NEURONAL CIRCUITRY OF ESTROGENIC EFFECTS ON THERMOREGULATION." Diss., The University of Arizona, 2010. http://hdl.handle.net/10150/195591.
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Approximately 75% of menopausal women in the United States experience hot flushes but the etiology of this thermoregulatory disorder is unknown. The dominant theory is that estrogen withdrawal sensitizes thermoregulatory areas of the brain, leading to the inappropriate activation of heat loss effectors in response to mild stimuli. This dissertation examines the circuitry of estrogen effects on thermoregulation. First, a rodent model was characterized. In ovariectomized rats, estradiol treatment decreased tail skin vasodilatation, a primary heat loss mechanism, and raised the ambient temperature threshold for tail skin vasodilatation. These results indicate that estradiol does not alter the maximal ability of blood vessels to constrict and dilate, but rather shifts the threshold for thermoregulatory activation in rats. Using this animal model, we examined how estradiol treatment and ambient temperature affect neuronal activity in brain areas involved with thermoregulation and reproduction. Out of 14 examined regions, only 3 areas were significantly affected by both estradiol and temperature and only the median preoptic nucleus (MnPO) exhibited increased activity at warmer ambient temperature. Interestingly, the effects of estradiol and ambient temperature on MnPO activity closely resembled their effects on tail skin vasodilatation. These results identify the MnPO as a plausible site for the integration of estrogen with skin vasodilatation. In the third study, we examined whether thermoregulation can be modified by neurokinin 3 (NK3) receptors, the dominant receptor for neurokinin B (NKB). Core temperature in ovariectomized rats was decreased by microinfusion of a selective NK3 receptor agonist into the MnPO and adjacent septal areas. This transient hypothermia was accompanied by a lack of homeostatic tail skin vasoconstriction but was not caused by tail skin vasodilatation or a global impairment in thermoregulation. These results demonstrate that thermoregulation in rats is modified by NK3 receptors in brain areas that receive projections from NKB neurons. In humans, menopause is associated with hot flushes and the hypertrophy and increased NKB gene expression in arcuate (infundibular) neurons. We propose a novel theory that estrogen withdrawal causes hot flushes by enhancing NKB release from arcuate (infundibular) neurons onto NK3 receptors.
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Murnin, Mark Anthony. "Structure-activity studies on some analogues of neurokinin A." Thesis, University of Ulster, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.260519.
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Wang, Linhong. "Differential processing of circulating substance P and neurokinin A /." The Ohio State University, 1995. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487862972134708.
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Shanab, Ahmed Ayesh Abu. "Synthesis and biological testing of some analogues of neurokinin A." Thesis, Queen's University Belfast, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.335458.
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Misu, Ryosuke. "Development of Neuropeptide Receptor Ligands for the Control of Reproductive Systems." 京都大学 (Kyoto University), 2015. http://hdl.handle.net/2433/199501.
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Wong, Chui Mae. "Substance P and neurokinin A as markers of pain in neonates." Thesis, University of Edinburgh, 2006. http://hdl.handle.net/1842/25329.
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Both substance P (SP) and neurokinin A (NKA) have been postulated to be involved in persistent pain in adult studies, but never previously researched in newborn infants. Methods of sample handling and analysis were developed to accommodate neonatal microsamples. Sample extraction was found to be essential, as were speed of sample collection and the use of polypropylene materials during sample handing. The clinical study enrolled 174 infants of different gestational ages, who had serial measurements of SP, NKA, and cortisol performed in plasma and saliva samples. Plasma SP concentrations in neonates ranged from 0.0-11.2 pmol/L (median 1.7 pmol/L) and NKA concentrations from 1.8-74.6 pmol/L (median 6.0 pmol/L). Gestation and birth weight had no significant correlation with peptide concentrations. Postnatally, there was a gradual rise in median plasma SP and NKA during the first three days which decreased again by days 7 to 14. Perinatal factors such as labour, the mode of delivery, and epidural analgesia affected NKA but not SP concentrations. With regard to either pain or assisted ventilation, plasma SP concentrations did not appear to be a useful marker of persistent pain or distress. Conversely, plasma NKA concentrations showed significant changes with ventilation, which were further modulated by the use of analgesia. Cortisol responses in the same group of infants demonstrated significant changes with ventilation, but not with the administration of analgesia. This suggests that although cortisol is a useful indicator of overall stress, NKA might be more specific for pain. There was a weak correlation between plasma SP and NKA. Plasma SP did not correlate with plasma cortisol or other physiological measures of pain used in this study.
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McLeod, Amber L. "Synaptic organization of the neurokinin system in the sensory spinal cord." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape9/PQDD_0026/NQ50219.pdf.
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Henderson, Alden Keith. "Characterization of the cloned neurokinin A receptor transfected in murine fibroblasts." Diss., The University of Arizona, 1992. http://hdl.handle.net/10150/185828.
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The neurokinins comprise a family of peptides with similar structure and physiological effects. Several limitations arise when investigating their biochemical and pharmacological properties. Because neurokinins contain similar amino acids in their carboxyl terminus, radiolabeled neurokinins and biosynthesized antibodies may lack the specificity to distinguish between the various neurokinin receptors. Tissue preparations used to characterize their pharmacological responses often have several types of neurokinin receptors, different receptor densities, or signal transduction mechanisms. These factors can complicate the interpretation of results from functional assays. Developing a cell line that expresses a high density of one type of neurokinin receptor with similar biochemical and pharmacological characteristics as the endogenous receptor may overcome these limitations. To facilitate this aim, the neurokinin-2 receptor (NK-2) was expressed by transfecting a bovine stomach cDNA that encoded for the NK-2 receptor into the murine fibroblast B82 L cell line. This de novo protein displayed high affinity and selectivity for neurokinin A (NKA) in competitive radioligand binding assays and was functionally coupled to phosphatidylinositide 4,5-bisphosphate (PIP₂) hydrolysis and intracellular calcium mobilization. The expressed receptor also exhibited a time dependent, homologous desensitization of PIP₂ hydrolysis. The rank order of affinity in competitive [¹²⁵I]NKA binding and potency for accumulation of [³H] inositol monophosphates ([³H]IP₁) in transfected B82 fibroblasts was NKA > substance P > senktide which classified the expressed protein as an NK-2 receptor. The expressed NK-2 receptors in B82 fibroblasts and the endogenous NK-2 receptors in bovine stomach had similar pharmacological properties. This implies that the in vitro expression system produced a protein whose pharmacological and biochemical characteristics resemble the endogenous receptor. The antagonists MEN 10207 and MEN 10208 blocked NKA-induced PIP₂ hydrolysis. In addition, the different pA₂ values of these peptides in transfected B82 cells, rabbit pulmonary artery, and hamster trachea of these peptides suggest NK-2 receptor heterogeneity in these tissues. The availability of a cell line expressing a homogeneous population of NK-2 receptors that mimics the phramacological characteristics of the endogenous receptor allows closer examination of ligand-receptor-effector interactions in a controlled environment. The novel reporting of an NK-2 receptor subtype and desensitization may provide a mechanism for intervention of physiological responses mediated by this receptor subtype.
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Menzies, John. "An investigation into the functional role of some neuropeptides in intestinal function with particular reference to inflammatory bowel disease and idiopathic chronic constipation." Thesis, Glasgow Caledonian University, 1999. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.301472.
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Bane, Steven Edward. "Expression and characterization of the human neurokinin 1 receptor from Escherichia coli." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 99 p, 2007. http://proquest.umi.com/pqdweb?did=1342742951&sid=1&Fmt=2&clientId=8331&RQT=309&VName=PQD.
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Hafizi, Sepehr. "Brain neurokinin-1 receptors in depression and interactions with the serotonin system." Thesis, University of Oxford, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.504364.
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Haley, Gwendolen E. "Contribution of neurokinin 3 receptor signaling to systemic vasopressin and oxytocin release." Laramie, Wyo. : University of Wyoming, 2008. http://proquest.umi.com/pqdweb?did=1594497031&sid=1&Fmt=2&clientId=18949&RQT=309&VName=PQD.
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Veser, Anika [Verfasser], and Achim [Akademischer Betreuer] Göpferich. "Multivalently binding antagonists for the neurokinin-1 receptor / Anika Veser ; Betreuer: Achim Göpferich." Regensburg : Universitätsbibliothek Regensburg, 2020. http://d-nb.info/1203874774/34.
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McCutcheon, James Edgar. "Genetic background influences the effect of neurokinin-1 receptor "knockout" in the mouse." Thesis, University College London (University of London), 2007. http://discovery.ucl.ac.uk/1444819/.
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Strains of mice used in laboratory research differ markedly at a molecular, anatomical and behavioural level. Genetic manipulation in mice has become a widely used tool for the selective study of single genes and has also produced a great diversity of phenotypes. However, the interaction between manipulated genes and background strain has been less well examined. Here, the effect of neurokinin-1 (NK1) receptor disruption is shown to depend heavily on the genetic background of the mouse that it is studied in. NK1_/_ mice have previously been shown to display an anxiolytic and antidepressant-like phenotype, as well as having reduced sensitivity to the rewarding properties of the opiates. Here, after being transferred onto a C57BL/6 (B6) background, the anxiolytic effect of the mutation is lost, as is the differential response to morphine's locomotor stimulating properties. When the mutation is transferred onto a mixed C57BL/6 x 129/sv (B6:129) background the NK1 /_ mouse becomes more sensitive to morphine than wildtype counterparts, although no difference in anxiolysis is evident between the genotypes. Examination of the HPA axis in these animals revealed that B6:129 animals had higher levels of stress-induced corticosterone release than B6. Furthermore, NK1 /_ animals on the B6:129 background expressed higher levels of hippocampal glucocorticoid receptor and exhibited more neurogenesis in the hippocampus than wildtype. No differences in these parameters were observed for the genotypes in the B6 strain. Comparison of CRF and cFos mRNAs did not reveal any differences between genotypes in either strain. These results show that there are important epistatic interactions between genetic background and the NK1 receptor. These findings may be relevant for the treatment of anxiety disorders and opiate addiction in the genetically heterogeneous patient population.
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Heppenstall, Paul Alexander. "Mechanisms of neurokinin₁ receptor action in the dorsal horn of the spinal cord." Thesis, University of Edinburgh, 1998. http://hdl.handle.net/1842/29799.
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This study addressed the role of neurokin1 (NK1) receptors in nociceptive transmission and their participation in a series of events involving glycine and NMDA receptor-mediated effects on spinal neurons. Using an in vivo electrophysiology protocol utilising ionosphoresis and extracellular recording from laminae III-V dorsal horn neurones of anaesthetised rats, the mechanisms of these interactions were assessed. The functions of the inflammatory cytokine leukaemia inhibitory factor (LIF) were also considered. Injury-induced alterations in the spinal expression pattern of this factor and the consequences of these changes to neuropeptide and excitatory amino acid expression were measured using in situ hybridisation. 1. The involvement of NK1 receptors in spinal pain transmission may be dependent upon the duration and intensity of the nociceptive stimulus. 2. NK1 receptors can contribute to the processing of sustained nociceptive stimuli by modulating excitatory amino acid-mediated transmission, particularly through potentiation of NMDA receptor activity. 3. LIF is a neuroactive cytokine that is associated with peripheral nerve injury. Using in situ hybridisation, the present study has examined the distribution of LIF mRNA in the spinal cord, normally or following peripheral inflammation or nerve injury and determined the consequences of intrathecally applied LIF on spinal expression of NK1 receptor and the high affinity glutamate transporter, EAAT2. In control animals, dorsal horn expression of LIF was high in superficial laminae I-II with only light expression in the deeper laminae III-V and in the ventral horn. Both peripheral inflammation and neuropathy significantly increased levels of LIF mRNA in the dorsal horn and this was most evident in the lateral parts of laminae I and II. Interactions within the spinal cord may underlie the plasticity of the dorsal horn in sensory processing. This has been discussed with reference to the regulation of short-term co-operation between NK1 and NMDA receptors by glycine and to long-term modifications of peptide and excitatory amino acid neurotransmission by altered LIF gene expression.
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Frick, Andreas. "Imaging Anxiety : Neurochemistry in Anxiety Disorders Assessed by Positron Emission Tomography." Doctoral thesis, Uppsala universitet, Institutionen för psykologi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-261983.
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Anxiety disorders, including social anxiety disorder (SAD) and posttraumatic stress disorder (PTSD) are common and disabling conditions. Largely based on animal and pharmacological studies, both the serotonergic and substance P/neurokinin-1 (SP/NK1) systems have been implicated in their underlying pathology. However, only few neuroimaging studies have directly assessed these neurotransmitter systems in human sufferers of anxiety disorders, and none have addressed possible between-systems relationships. The overall aim of this thesis was to study possible neurochemical alterations associated with anxiety disorders. To this end, three studies using positron emission tomography (PET) for in-vivo imaging of the brain serotonergic and SP/NK1 systems in patients with SAD and PTSD were conducted. The radiotracers [11C]5-HTP, [11C]DASB, and [11C]GR205171 were used to index serotonin synthesis rate, serotonin transporter (SERT) availability, and NK1 receptor availability respectively. In Study I, patients with SAD relative to controls exhibited enhanced serotonin synthesis rate and serotonin transporter availability. Serotonin synthesis rate in the amygdala was positively related to social anxiety symptom scores. Study II demonstrated increased NK1 receptor availability in the amygdala in patients with SAD relative to controls. In Study III, patients with PTSD showed elevated NK1 receptor availability in the amygdala as compared to controls. SERT availability in the amygdala was negatively related to PTSD symptom severity, a relationship that was moderated by NK1 receptor levels. The regional overlap between SERT and NK1 receptor expression was altered in patients with PTSD, with reduced overlap linked to more severe symptoms. Collectively, the findings are consistent with the view that serotonin in the amygdala induces rather than reduces anxiety and links exaggerated anxiety to an overactive presynaptic serotonin system. In addition, the involvement of the SP/NK1 system in stress and anxiety, as suggested by animal studies, was demonstrated in two common human anxiety disorders. Finally, PTSD symptomatology is better accounted for by interactions between the serotonergic and SP/NK1 systems in the amygdala than by each system separately. In conclusion, this thesis supports that both the serotonergic and SP/NK1 systems in and of themselves, but also interactively, may be important contributors to anxiety symptomatology.
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Skinner, David P. "Hormone Induced "Migraine" and Attempts at Blocking Opiate Reward through NK1." Diss., The University of Arizona, 2014. http://hdl.handle.net/10150/321547.
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Migraine headache is one of the most common neurological disorders. While the mechanisms contributing to migraine pathophysiology have yet to be fully elucidated, the disproportionate number of post-pubescent, pre-menopausal women affected suggests a central role for female hormones, such as estrogen. The mechanism(s), however, by which estrogen contributes to migraine have yet to be deciphered. Cortical spreading depression (CSD) is associated with "Classic Migraine", now referred to as migraine with aura. Here we use a well-established animal model for migraine with aura to test the putative role of estrogen in the development of CSDs in awake and freely moving female rats. Beta estradiol administration in ovariectomized female rats resulted in a significant increase in CSD episodes over a 12-hour recording period. Additionally, beta estradiol administration in these rats promoted migraine-associated behavior, significantly reducing exploratory behavior (i.e., number of vertical rearing episodes) when compared to vehicle-treated controls. Critically, the increase in CSD episodes was completely abolished with pre-administration of ICI 182,780 a pure alpha and beta estrogen receptor antagonist. ICI 182,780administration also blocked beta estradiol-induced migraine-associated behaviors, restoring vertical rearing episodes to baseline levels. These data illustrate that an increase in estrogen levels in an animal that no longer produces estrogen (postmenopausal characteristic) can promote the development of CSDs. These data suggest that an estrogen receptor-mediated mechanism may drive episodes of migraine with aura and highlight the need for further investigation into estrogen's role in migraine.
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Slone-Murphy, J. "Behavioural and molecular responses to amphetamine in the neurokinin-1 receptor knock-out mouse." Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1302406/.
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The neurokinin-1 receptor knock-out (NK1R-/-) mouse is hyperactive and shows deficits in attentional processing, and has recently been put forward as a model of attention deficit hyperactivity disorder (ADHD). Acute amphetamine, a first-line treatment for ADHD and a drug of abuse, paradoxically reduces the hyperactivity of NK1R-/- mice, and the characteristic amphetamine-stimulated increase in striatal dopamine efflux seen in wild-type animals is attenuated in NK1R-/- mice. The research presented in this thesis centres on the behavioural and molecular responses of NK1R-/- mice to amphetamine. Over the course of this work, a fundamental deficit in the striatal cholinergic neuroanatomy of NK1R-/- and wild-type mice was discovered. While the responses of NK1R-/- mice to amphetamine were normal compared with wild-types, NK1R-/- mice were found to have a reduced number of cholinergic interneurones in the lateral striatum, independent of treatment, and an increased number of cholinergic neurons in the nucleus basalis. In the striatum, the reduction in cell numbers was specific to cholinergic interneurones, which all express the NK1 receptor in wild-type animals and play a key role in regulating striatal dopamine release. The number of striatal parvalbumin-expressing interneurones, which do not express NK1 receptors, did not differ between NK1R-/- and wildtype mice. The present results demonstrate that global disruption of the NK1 receptor results in significant alterations in central cholinergic system neuroanatomy, which may contribute to the previously observed impaired dopaminergic response to amphetamine. The results presented in this thesis provide support for emerging evidence that deficits in cholinergic transmission play a role in the pathophysiology of ADHD, and could have significant implications for future ADHD research.
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Dudley, J. A. "Evaluation of the neurokinin-1 receptor knockout mouse model of Attention Deficit Hyperactivity Disorder." Thesis, University College London (University of London), 2013. http://discovery.ucl.ac.uk/1390670/.
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Attention Deficit Hyperactivity Disorder (ADHD) is a common neurobehavioural disorder in children and often persists into adulthood. Current treatments lack efficacy in a large number of patients, and the aetiology of ADHD needs to be further elucidated in order to develop alternative treatments. The neurokinin-1 receptor knockout (NK1R-/-) mouse has recently been proposed as an animal model of ADHD; it is hyperactive and this is reduced by psychostimulants, polymorphisms in the TACR1 gene (the human NK1R gene) have been found in patients with ADHD, and NK1R-/- mice have deficits in monoamine transmission, consistent with the pathophysiology of ADHD. Finally, the NK1R-/- mouse exhibits inattentiveness, impulsivity and perseveration in the 5-Choice Serial Reaction-Time Task (5-CSRTT). The aim of this thesis was to further validate the NK1R-/- mouse model of ADHD by studying the effects of d-amphetamine on its performance in the 5-CSRTT, and subsequently to test alternative drugs that could be therapeutically beneficial. However, over the course of this work, the hyperactivity of NK1R-/- mice was not stable when tested acutely. Therefore, telemetry was used to monitor the activity of mice in their homecage over many days. Also, the breeding method was expanded to include heterozygous matings in order to minimise genetic drift. Whilst hyperactivity in this test was robust in both colonies, the impulsivity seen in NK1R /- mice bred from homozygous matings was not present in NK1R-/- mice bred from heterozygotes. Furthermore, there was a reduction in choline acetyltransferase-positive cells in the striatum of homozygously-bred but not heterozygously-bred NK1R-/- mice. These findings highlight important interactions between early environment, NK1R function and the central cholinergic system. They also support evidence suggesting that abnormal cholinergic transmission is involved in ADHD, and could underlie impulsivity. Finally, these findings could have important implications for future research on impulse control disorders.
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Garnier, Agnès [Verfasser], and Roland [Akademischer Betreuer] Kappler. "Neurokinin-1 receptor as a therapeutic target in hepatoblastoma / Agnès Garnier ; Betreuer: Roland Kappler." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2016. http://d-nb.info/1123957282/34.
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Largent-, Milnes Tally Marie. "NEUROKININ 1 RECEPTORS AND THEIR ROLE IN OPIOID-INDUCED HYPERALGESIA, ANTINOCICEPTIVE TOLERANCE AND REWARD." Diss., The University of Arizona, 2010. http://hdl.handle.net/10150/193763.
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Pain is the most common and debilitating sign of a medical problem, with nearly 15 million patients suffering from chronic pain, including neuropathic pain. Widely used therapies for treating neuropathic pain include tri-cyclic antidepressants, opioids, anticonvulsants, non-steroidal anti-inflammatory agents and combinations thereof. Despite the abundance of treatments, the management of chronic pain remains difficult due to an inability for many patients to achieve appropriate pain relief at doses which are tolerable over long periods of time.Opiates (natural products), or opioids (synthetic derivatives), are considered the gold standard of analgesic care, though with little efficacy for neuropathic pain. Opioids are associated with unwanted side effects, including paradoxical pain and abuse liability that may result from several nervous system adaptations within the pain modulating neural network. These dose related side effects become more prevalent as clinicians try to overcome analgesic tolerance.Molecular mechanisms underlying these unwanted side effects have been studied extensively, and the literature purports a variety of contributing factors and neurobiological adaptations. The studies herein describe additional molecular adaptations and novel pharmacological approaches to counteract these changes. First, the contributions of neurobiological remodeling within a single receptor system (the opioid system) were investigated in the spinal dorsal horn after peripheral nerve ligation and chronic exposure to an opioid agonist in combination with an ultra-low-dose of opioid antagonist. The effects of the ultra-low-dose opioid antagonist naltrexone on the efficacy of oxycodone for neuropathic pain were investigated after both central and systemic administration.Secondly, molecular remodeling occurs across different receptor systems in the pain network, including altered regulation of pronociceptive molecules (e.g. substance P; SP). Previous studies have reported that opioid-induced hyperalgesia, tolerance and reward can be prevented by a blockade or ablation of SP activity at the neurokinin 1 receptor (NK1). We have characterized single compounds, rationally designed to act as opioid agonists and an NK1 antagonist using in vitro assays and the efficacy in vivo using rodent models of pain, antinociceptive tolerance and reward. Collectively, these studies validate the concept of targeting multiple neurobiological adaptations as a therapeutic option for neuropathic pain and reducing opioid- mediated side effects.
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Skorupskaite, Karolina. "Kisspeptin and neurokinin B in the regulation of the human hypothalamic-pituitary-gonadal axis." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/28897.
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Background: Hypothalamic kisspeptin and neurokinin B (NKB) are central regulators of GnRH and thus gonadotropin (LH and FSH) secretion. Men and women with loss-of-function mutations in NKB-kisspeptin pathway show hypogonadotropic pubertal delay with reduced GnRH/LH pulsatility. Studies in patients with defects in NKB signalling suggest that kisspeptin is functionally downstream of NKB, although there are very limited data on the relevance of the NKB pathway in normal men or women, and no hierarchical data on this. The studies described in this thesis have investigated the interaction between these neuropeptides in the control of human reproduction in conditions of varying sex-steroid environment, and in states of fast and slow LH secretion (men, menopause, various stages across the menstrual cycle). Overall hypothesis: Pharmacological blockade of NKB signalling will decrease LH secretion by modulating GnRH/LH pulsatility, indicating the involvement of the NKB pathway in normal human reproductive function. It is also hypothesised that this will not abrogate the stimulatory kisspeptin response, revealing a functional hierarchy whereby NKB signalling is upstream of kisspeptin. Research strategy: A specific neurokinin-3 receptor antagonist (NK3R antagonist, AZD4901) was administered 40 mg twice daily orally for 7 days with and without kisspeptin-10 (KP-10) challenge. Response of reproductive hormones (serum and urinary where applicable) was measured. LH was sampled every 10 minutes for 8 hours to assess LH pulsatility by blinded deconvolution. Results: Role of neurokinin B and kisspeptin in healthy men Six healthy men underwent LH pulsatility study pre-treatment and on day 7 of NK3R antagonist administration with iv KP-10 bolus (0.3 μg/kg) at 6 hours. NK3R antagonist reduced LH and testosterone secretion, whilst stimulatory LH response to KP-10 was unaffected. LH pulse frequency was unchanged by the NK3R antagonist but basal (nonpulsatile) and pulsatile LH secretion was markedly reduced. Role of neurokinin B and kisspeptin in postmenopausal women Eleven postmenopausal women underwent LH pulsatility study pre-treatment and on day 7 of NK3R antagonist administration with iv KP-10 bolus (0.3 μg/kg) at 6 hours. NK3R antagonist decreased LH secretion. Basal (nonpulsatile) LH secretion also fell and while LH pulse frequency did not change in a group as a whole, it did fall in the 8 of 11 postmenopausal womenwith hot flushes. These women reported a reduction in hot flush frequency (3.4±1.2 vs 1.0± 0.6 flushes/day with NK3Ra, p=0.008) and severity whilst on NK3R antagonist. LH response to KP-10 was minimal and unaffected by the NK3R antagonist. Role of neurokinin B across different phases of menstrual cycle The effect of NK3R antagonist on ovarian function was compared in early follicular (n=13), late follicular (n=6) and luteal phase (n=6) to no treatment control cycle. Early follicular: NK3R antagonist was commenced from cycle day 5-6. The diameter of the leading follicle was smaller than in controls at the end of treatment (9.3±0.4 vs 15.1±0.9 mm, p < 0.0001). Serum estradiol was also reduced and the endometrium was thinner. Although NK3R antagonist had no effect on LH pulse frequency, basal (nonpulsatile) LH secretion was decreased, suggesting that NKB modulates GnRH secretion. After stopping treatment, follicle development resumed and estradiol secretion increased thereby delaying the LH surge in 11/13 women (LH surge cycle day 22±1 vs 15±1, p=0.0006). The delayed LH surge and ovulation were confirmed by a similarly delayed rise in urinary progesterone and prolonged cycle length. NK3R antagonist did not affect luteal function. Late follicular: NK3R antagonist was administered from the emergence of a dominant follicle (≥12mm). Whilst there was an LH surge in all treated cycles, estrogen feedback was perturbed by the NK3R antagonist, as there was increased variation in the timing of LH surge compared to control cycle. NK3R antagonist had no effect on the growth of a dominant follicle and luteal function was unaffected. Luteal: NK3R antagonist was administered from day +2-3 of the disappearance of the dominant follicle. NK3R antagonist reduced the variation in the timing of peak estradiol secretion. Estradiol and progesterone concentrations remained unchanged, suggesting that luteal function was overall unaffected by this treatment. No difference in mean LH was observed, although LH pulsatility was not assessed. Role of neurokinin B and kisspeptin in the mid-cycle LH surge A model of follicular phase (cycle day 9-11) administration of estradiol (200μg/day) to induce LH secretion at 48 hours was used in twenty women, mimicking LH surge. In this model, KP-10 infusion (4μg/kg/hr for 7 hours) enhanced LH secretion, the response of which was directly correlated with estrogen concentration, indicating a role of kisspeptin in estrogen feedback. Pre-treatment with NK3R antagonist decreased LH pulse frequency and whilst the immediate LH response to KP-10 was unaffected, it blunted the duration of this response and abolished the relationship between estradiol and kisspeptin-induced LH secretion. Conclusions: These data indicate the role of NKB-KP pathway in regulating human reproductive function and that this is via the modulation of pulsatile GnRH secretion. Whilst NKB is predominantly proximal to kisspeptin, the hierarchy is more complex than simply linear in the control of human HPG axis. Manipulation of NKB-KP signalling has therapeutic potential in regulating GnRH/LH secretion in wide range of clinical settings, including contraception, sex-steroid dependent disorders and in the treatment of hot flushes.
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Yamamoto, Koki. "Structure-activity Relationships for Development of Neurokinin-3 Receptor Antagonists with Reduced Environmental Impact." Kyoto University, 2019. http://hdl.handle.net/2433/242671.
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Cholanian, Marina. "Effects of Estrogen on Morphological and Electrophysiological Properties of Arcuate NKB Neurons." Diss., The University of Arizona, 2013. http://hdl.handle.net/10150/311477.
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Infundibular (arcuate) neurokinin B (NKB) neurons play a critical role in neuroendocrine control of reproduction. Specifically, a local network of arcuate neurons that co-express kisspeptin, neurokinin B, and dynorphin (so-called, KNDy neurons), has emerged as a potential pacemaker driving the pulsatile secretion of gonadotropin-releasing hormone (GnRH) that is required for normal reproduction. These neurons are the target of estrogen and may be an important link in estrogen negative feedback on GnRH functioning. KNDy neurons respond to estrogen withdrawal with dramatic changes in gene expression and somatic hypertrophy, an effect that is reversible by estradiol replacement. Studies addressing the effects of estrogen withdrawal and replacement on morphological and electrophysiological features of KNDy neurons have been hindered by the inability to target this subpopulation of neurons in the live tissue. This dissertation examines estrogen-induced changes in arcuate NKB circuitry and excitability and discusses its implications in reproductive axis. First, the novel Tac2-EGFP transgenic mouse model was characterized. The reproductive function, EGFP-ir distribution in the brain, and co-localization of EGFP with proNKB in the arcuate nucleus were examined and compared to littermate controls. Indices of reproductive function (puberty onset, estrous cyclicity, and LH pulsatility) were comparable between Tac2 and wildtype mice, suggesting that the transgenic animals have preserved estrogen negative feedback. The long-term estrogen withdrawal via ovariectomy and estradiol replacement model was used to examine electrophysiological and morphological changes in arcuate NKB neurons. We found that low-dose chronic estradiol replacement results in decreased excitability of arcuate NKB neurons, a finding that is consistent with the proposed role of this neuronal population in estrogen negative feedback on reproductive axis. Changes in excitability were seen despite the overall similarity in intrinsic properties of estradiol-treated and untreated ovariectomized mice. We also demonstrated for the first time that single arcuate NKB neurons form a local network by way of recurrent collaterals. Axonal targets of single NKB neurons included the internal zone of the median eminence, ependymal layer of the 3rd ventricle, and sites lateral and dorsal to the borders of the arcuate nucleus. Long-term treatment with estradiol resulted in decreased somatic volume and decreased dendritic spine density. Together, these data demonstrate that low-dose chronic estradiol replacement in ovariectomized mice resulted in morphological plasticity of arcuate NKB neurons that was accompanied by changes in excitability of this neuronal population, supporting the role of these neurons in estrogen negative feedback on GnRH secretion.
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Prüfer, Thomas. "Ischämie-Reperfusionsschäden in Fettlebern : Aggravierung durch Resektion und Prävention durch Glutathion und Neurokinin-1 Rezeptorblockade /." München, 2008. http://opac.nebis.ch/cgi-bin/showAbstract.pl?sys=000254364.
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Palmer, James Alexander. "The expression and regulation of the neurokinin-1 receptor in the rat central nervous system." Thesis, University of Cambridge, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.627046.
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Nwaneshiudu, Chinwe A. "Characterization of a functional role of the neurokinin-3 receptor in behavioral effects of cocaine." Diss., Temple University Libraries, 2011. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/62614.
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PharmacologyPh.D.
The tachykinin NK-3 receptor is a G-protein coupled receptor activated by mammalian tachykinin neuropeptides, which can modulate dopaminergic neurotransmission, and alter dopamine-mediated behaviors. The NK-3 receptor is currently under investigation as a novel therapeutic target for cocaine addiction. Our studies, as outlined in this dissertation, sought to determine if NK-3 receptors have a functional role in the acute as well as long-term behavioral effects of cocaine. Administration of NK-3 receptor agonists or antagonists potentiates or attenuates dopamine-mediated behaviors, respectively. Based on these findings, we hypothesized that blockade of neurokinin-3 receptors would alter acute and long-term behavioral responses to cocaine. We investigated whether acute and repeated administration of the NK-3 receptor antagonist SB 222200 altered hyperactivity induced by cocaine, and determined a possible mechanism involving dopamine D1 receptors in the striatum. We also determined whether NK-3 receptor blockade altered the development and expression of behavioral sensitization after repeated cocaine administration. Lastly, we investigated whether modulation of behavioral effects of acute and repeated cocaine by NK-3 receptors involved GSK3 phosphorylation in the nucleus accumbens. As described in this dissertation, we show that acute administration of the NK-3 receptor antagonist SB 222200 before a cocaine injection attenuated stereotypic responses produced by cocaine. Repeated administration of SB 222200 enhanced stereotypic activity produced by either cocaine or a low dose of SKF 82958 (0.125 mg/kg, i.p.) when administered seven days later. Dopamine receptor binding studies were performed to determine the mechanism of enhanced stereotypic responses. Binding studies showed a 19.7% increase in dopamine D1 receptor density in the striatum seven days later after repeated SB 222200 administration. These findings demonstrate that acute blockade of NK-3 receptors attenuated cocaine-induced behaviors in agreement with previous studies. Furthermore, these studies also show novel effects of repeated blockade of NK-3 receptors, which causes subsequent enhancement of cocaine and dopamine D1 receptor-mediated behaviors, possibly resulting from dopamine D1 receptor up-regulation in the striatum. In order to determine a role of NK-3 receptors in the development of cocaine-induced behavioral sensitization, the NK-3 receptor antagonist SB 222200 (2.5 or 5 mg/kg, s.c.) was administered prior to daily cocaine injections for 5 days. After a 7-day drug-free period, behavioral responses to a cocaine challenge were measured. Repeated administration of cocaine for 5 days induced a sensitized response upon a cocaine challenge 7 days later. Administration of SB 222200 prior to daily cocaine attenuated the development of behavioral sensitization. Moreover, administration of SB 222200 prior to the cocaine challenge blocked the expression of behavioral sensitization. These findings demonstrate that NK-3 receptor activity is involved in the development and expression of behavioral sensitization to cocaine. Lastly, we examined GSK3 phosphorylation in the nucleus accumbens induced by acute and repeated cocaine administration and determined if phosphorylation was altered by NK-3 receptor blockade. Similar to the drug administration regimens used in the behavioral studies, the NK-3 receptor antagonist SB 222200 was administered 30 mins prior to an acute cocaine injection. The nucleus accumbens was examined for changes in GSK3 phosphorylation by Western blot analysis. Increases in phosphorylation of the isoforms, GSK3α and GSK3β in the nucleus accumbens were detected 20 mins after an acute injection of cocaine. NK-3 receptor blockade prior to cocaine administration did not alter the cocaine-induced increase in GSK3 phosphorylation. Similar to the behavioral sensitization studies, SB 222200 was administered prior to repeated cocaine for 5 days, and 7 days later GSK3 phosphorylation was measured after a subsequent cocaine challenge. In contrast to the increases in GSK3α and GSK3β in the nucleus accumbens after an acute cocaine injection, no regulation of GSK3 phosphorylation was found after prior repeated cocaine administration and cocaine challenge. Administration of SB 222200 prior to repeated cocaine produced an increase in GSK3α and GSK3β phosphorylation after a cocaine challenge. Collectively, these data point to involvement of NK-3 receptor activity in changes in the phosphorylation of GSK3 in the nucleus accumbens produced by cocaine. In summary, functional involvement of NK-3 receptors in acute and long-term behavioral effects of cocaine was investigated. In agreement with previous findings, studies in this dissertation demonstrate that acute blockade of NK-3 receptors attenuates cocaine-induced behaviors. In addition, we found novel effects of repeated blockade of NK-3 receptors on cocaine-induced hyperactivity. There is enhancement of subsequent cocaine and dopamine D1 receptor-mediated behaviors possibly due to dopamine D1 receptor up-regulation in the striatum. NK-3 receptor activity was shown to be involved in long-term behavioral effects of cocaine and molecular changes in GSK3 phosphorylation in the nucleus accumbens. Blockade of NK-3 receptors prevented the development and expression of behavioral sensitization to cocaine and also blocked the changes in the phosphorylation of GSK3 in the nucleus accumbens. This dissertation has demonstrated a role of NK-3 receptors in modulating acute as well long-term cocaine-induced behavioral hyperactivity. Therefore, there is potential clinical relevance of NK-3 receptors in cocaine abuse and dependence as a therapeutic target for treatment, which warrants further characterization in future preclinical and clinical investigations.
Temple University--Theses
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Makeham, John Murray. "Functional neuroanatomy of tachykinins in brainstem autonomic regulation." University of Sydney, 1997. http://hdl.handle.net/2123/1960.
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Doctor of Philosophy (PhD)Little is known about the role that tachykinins, such as substance P and its receptor, the neurokinin-1 receptor, play in the generation of sympathetic nerve activity and the integration within the ventrolateral medulla (VLM) of many vital autonomic reflexes such as the baroreflex, chemoreflex, somato-sympathetic reflex, and the regulation of cerebral blood flow. The studies described in this thesis investigate these autonomic functions and the role of tachykinins through physiological (response to hypercapnoea, chapter 3), anatomical (neurokinin-1 receptor immunohistochemistry, chapter 4) and microinjection (neurokinin-1 receptor activation and blockade, chapters 5 and 6) experiments. In the first series of experiments (chapter 3) the effects of chemoreceptor activation with hyperoxic hypercapnoea (5%, 10% or 15% CO2 in O2) on splanchnic sympathetic nerve activity and sympathetic reflexes such as the baroreflex and somato-sympathetic reflex were examined in anaesthetized rats. Hypercapnoea resulted in sympatho-excitation in all groups and a small increase in arterial blood pressure in the 10 % CO2 group. Phrenic nerve amplitude and phrenic frequency were also increased, with the frequency adapting back to baseline during the CO2 exposure. Hypercapnoea selectively attenuated (5% CO2) or abolished (10% and 15% CO2) the somato-sympathetic reflex while leaving the baroreflex unaffected. This selective inhibition of the somato-sympathetic reflex while leaving the baroreflex unaffected was also seen following neurokinin-1 receptor activation in the rostral ventrolateral medulla (RVLM) (see below). Microinjection of substance P analogues into the RVLM results in a pressor response, however the anatomical basis for this response is unknown. In the second series of experiments (chapter 4), the distribution of the neurokinin-1 receptor in the RVLM was investigated in relation to catecholaminergic (putative sympatho-excitatory “C1”) and bulbospinal neurons. The neurokinin-1 receptor was demonstrated on a small percentage (5.3%) of C1 neurons, and a small percentage (4.7%) of RVLM C1 neurons also receive close appositions from neurokinin-1 receptor immunoreactive terminals. This provides a mechanism for the pressor response seen with RVLM microinjection of substance P analogues. Neurokinin-1 receptor immunoreactivity was also seen a region overlapping the preBötzinger complex (the putative respiratory rhythm generation region), however at this level a large percentage of these neurons are bulbospinal, contradicting previous work suggesting that the neurokinin-1 receptor is an exclusive anatomical marker for the propriobulbar rhythm generating neurons of the preBötzinger complex. The third series of experiments (chapter 5) investigated the effects of neurokinin-1 receptor activation and blockade in the RVLM on splanchnic sympathetic nerve activity, arterial blood pressure, and autonomic reflexes such as the baroreflex, somato-sympathetic reflex, and sympathetic chemoreflex. Activation of RVLM neurokinin-1 receptors resulted in sympatho-excitation, a pressor response, and abolition of phrenic nerve activity, all of which were blocked by RVLM pre-treatment with a neurokinin-1 receptor antagonist. As seen with hypercapnoea, RVLM neurokinin-1 receptor activation significantly attenuated the somato-sympathetic reflex but did not affect the sympathetic baroreflex. Further, blockade of RVLM neurokinin-1 receptors significantly attenuated the sympathetic chemoreflex, suggesting a role for RVLM substance P release in this pathway. The fourth series of experiments (chapter 6) investigated the role of neurokinin-1 receptors in the RVLM, caudal ventrolateral medulla (CVLM), and nucleus tractus solitarius (NTS) on regional cerebral blood flow (rCBF) and tail blood flow (TBF). Activation of RVLM neurokinin-1 receptors increased rCBF associated with a decrease in cerebral vascular resistance (CVR). Activation of CVLM neurokinin-1 receptors decreased rCBF, however no change in CVR was seen. In the NTS, activation of neurokinin-1 receptors resulted in a biphasic response in both arterial blood pressure and rCBF, but no significant change in CVR. These findings suggest that in the RVLM substance P and the neurokinin-1 receptor play a role in the regulation of cerebral blood flow, and that changes in rCBF evoked in the CVLM and NTS are most likely secondary to changes in arterial blood pressure. Substance P and neurokinin-1 receptors in the RVLM, CVLM and NTS do not appear to play a role in the brainstem regulation of tail blood flow. In the final chapter (chapter 7), a model is proposed for the role of tachykinins in the brainstem integration of the sympathetic baroreflex, sympathetic chemoreflex, cerebral vascular tone, and the sympatho-excitation seen following hypercapnoea. A further model for the somato-sympathetic reflex is proposed, providing a mechanism for the selective inhibition of this reflex seen with hypercapnoea (chapter 3) and RVLM neurokinin-1 receptor activation (chapter 5). In summary, the ventral medulla is essential for the generation of basal sympathetic tone and the integration of many vital autonomic reflexes such as the baroreflex, chemoreflex, somato-sympathetic reflex, and the regulation of cerebral blood flow. The tachykinin substance P, and its receptor, the neurokinin-1 receptor, have a role to play in many of these vital autonomic functions. This role is predominantly neuromodulatory.
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Cain, James Patrick. "Design, Synthesis, and Evaluation of New Ligands for G Protein-Coupled Receptors and Kinases." Diss., The University of Arizona, 2011. http://hdl.handle.net/10150/204272.
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Peptidergic G Protein-Coupled Receptors (GPCRs) play a role in many of the most important biological functions, and the ability to modulate the activity of these critical proteins has tremendous potential to increase our understanding of biology and allow the development of new therapeutics. In some cases this knowledge will point towards the importance of interconnected proteins of the same or different classes, such as kinases, which interact in a complex and dynamic network in vivo. Understanding these systems will be crucial for addressing unmet therapeutic needs, and new chemical structures may be important at every step of the process.Our contribution to this pursuit includes the development of new ligands for the melanocortin receptors based on a bicyclic or tricyclic core structure. These were designed to be peptidomimetics, built from amino acids to leverage the accumulated knowledge of the group but with properties that complement those of peptides. Most of the molecules in this series bind to the melanocortin receptors, and many with significant selectivity. Some are selective for the MC5R, which may allow further study of this widely distributed but largely unexplored subtype. Others bind preferentially to the MC1R, a property which may be useful in the development of imaging agents targeting melanoma.Imaging using fluorescent probes can provide a tremendous amount of information in studies of receptor biology. With this in mind, we have developed new fluorescent ligands which bind to melanocortin receptors. These compounds use the previously discovered bicyclic template and incorporate the small organic fluorophores anthranilate and N-methylanthranilate.While these structures are in a sense bifunctional, as they exhibit both pharmacologic and fluorescent activity, other molecules may instead incorporate two different pharmacophores. We have synthesized designed multiple ligands (DMLs) of this type for the opioid and neurokinin receptors, as well as molecules which target both the opioid receptors and p38 MAP kinase. These structures merged known active ligands, such as fentanyl for the opioid activity, into one bifunctional molecule. In addition we have used our newly developed template to create a novel NK1R antagonist which may be part of the next generation of bifunctional ligands.
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Butcher, James William. "The nucleus tractus solitarii and cardiorespiratory control : the role of neurokinin-1 receptors and potassium channels." Thesis, University of Bristol, 1998. http://hdl.handle.net/1983/588f7506-6822-4a28-9a43-cf75ca1e375e.
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Chan, Wing Sai. "Roles of neurokinin receptor one in six-hydroxydopamine-lesioned rat : an animal model of Parkinson's disease." HKBU Institutional Repository, 2006. http://repository.hkbu.edu.hk/etd_ra/712.
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Gadd, Christopher Andrew. "The relationship of the neurokinin-1 receptor to reward and learning and memory behaviours in the mouse." Thesis, University College London (University of London), 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.404662.
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Sandweiss, Alexander Jordan, and Alexander Jordan Sandweiss. "The Role of Substance P in Opioid Induced Reward." Diss., The University of Arizona, 2016. http://hdl.handle.net/10150/621568.
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Chronic pain affects approximately 100 million Americans. Opioids are the mainstay therapy for the treatment of chronic pain. While physicians and patients alike are apprehensive about using opioids due to their side effects including respiratory depression and addiction, 259 million opioid prescriptions were written in 2012. Although opioids are the most efficacious available analgesics, they increase both positive and negative reinforcement, ultimately leading to addiction. The pro-nociceptive neurotransmitter, Substance P (SP) and its corresponding receptor (NK₁R), are not only found on pain pathways to promote pain but also found in the ventral tegmental area associated with dopamine neurons. Studies have shown that Substance P can potentiate positive reinforcement of opiates and may play a role in opioid reward. Here using in vivo microdialysis, we show that systemic morphine significantly increases SP release in the VTA, an effect mediated by ventral midbrain GABAergic neurons. Substance P administered to the VTA results in a significant increase in dopamine release in the nucleus accumbens (NAc). Using CRISPR-Cas9 knockdown of NK₁R in the VTA we prevent the induction of opiate reward as tested using a conditioned place preference paradigm (CPP). Finally, we developed a novel opioid agonist/NK₁R antagonist bifunctional compound, TY032, which inhibits acute and chronic pain in male rats. Importantly, TY032 microinjection into the VTA did not increase extracellular dopamine release in the NAc and did not produce a positive CPP score. These data indicate dual targeting of the dopamine reward circuitry and pain pathways with multifunctional opioid-NK₁R compounds may be an effective strategy in developing future analgesics that lack the potential for abuse.
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Barnard, Amanda Leann. "The Effects of KNDy Neuron Peptides on Prolactin and Luteinizing Hormone in Pup-Deprived Lactating Rats." OpenSIUC, 2014. https://opensiuc.lib.siu.edu/theses/1508.
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Lactation is the final stage of reproduction in mammals and is characterized by chronically elevated prolactin and suppressed luteinizing hormone. The neuroendocrine regulation of prolactin and luteinizing hormone during lactation are not fully understood. In the hypothalamic arcuate nucleus is a population of neurons known as KNDy neurons because they co-express the neuropeptides Kisspeptin, Neurokinin B and Dynorphin. These neurons are known to project to gonadotropin-releasing hormone cell bodes in the preoptic area and nerve terminals in the median eminence, which regulate the secretion of luteinizing hormone, and to dopaminergic tuberoinfundibular neurons in the arcuate nucleus, which are known to regulate prolactin. Because KNDy neurons project to neuronal populations known to regulate both prolactin and luteinizing hormone, the general hypothesis for these studies is that neuropeptides Kisspeptin, Neurokinin B and Dynorphin play a role in regulating these hormones or are regulated by these hormones during lactation. In a model of lactating rats deprived of their pups for 24 hours, intracerebroventricular injection of an endogenous Kisspeptin receptor ligand, Kp-10, modestly increased prolactin secretion and markedly increased luteinizing hormone secretion. Neither Neurokinin B nor the Neurokinin B receptor agonist, Senktide, had a significant effect on either hormone in this rat model. Dynorphin and U-50,488, a kappa opioid receptor agonist, robustly increased prolactin although no changes were measured in luteinizing hormone levels. In this model of 24-hour pup-deprived lactating rats, prolactin was responsive to kappa opioid receptor agonists and luteinizing hormone was responsive to Kisspeptin receptor agonists. In a second set of experiments, sense and anti-sense in situ hybridization probes were developed for Kiss1, the Kisspeptin gene, and Tac2, the gene encoding Neurokinin B. It was confirmed that the cDNA sequences cloned for these mRNAs were correct and were highly homologous to published rat mRNA sequences. In situ hybridization was performed using the Kiss1 and Tac2 probes, as well as a probe for Pdyn, which encodes Dynorphin. No specific cytoplasmic signal was observed using any of the three sense probes. With the anti-sense probes, clusters of reduced silver grains representing Kiss1, Tac2 and Pdyn mRNAs were observed in the arcuate nucleus, lateral to the third ventricle and superior to the median eminence. These expression patterns were consistent with the published literature. Also, the expression patterns for all three neuropeptides were similar to each other, suggesting that many of the arcuate nucleus neurons lateral to the third ventricle and superior to the median eminence are KNDy neurons.
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Wagner, Sabine. "Tachykinine und Tachykinin-Rezeptoren in der Innervation der Lunge der Maus : Veränderungen bei Hypoxie und BDNF-Überexpression /." Giessen : Köhler, 2004. http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&doc_number=014610775&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA.
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Svensson, Alexandra. "Immune regulation of herpes simplex virus type 2 infection : special emphasis on the transcription Factor T-bet /." Göteborg : Department of Rheumatology and Inflammation Research, The Sahlgrenska Academy, Göteborg University, 2006. http://hdl.handle.net/2077/852.
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Brayley, Kerensa. "Processing of the tachykinin precursor TAC3 in the human placenta and characterisation of the peptide product, neurokinin B (NKB)." Thesis, University of Reading, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.485703.
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Excessive secretion of the peptide, neurokinin B (NKB) into the circulation during the third trimester of pregnancy has been reported in women with the gestational disease, pre-eclampsia (PE). Therefore, the objective of this work was to develop a clinic friendly two-site enzyme-linked immunosorbent assay (ELISA) to detect levels of NKB directly from plasma samples of pregnant women and to determine the mechanism's of precursor processing. !t was hoped to confIrm the link between elevated levels of this peptide and PE. Initial assays of pregnancy plasmas using the two-site NKB ELISA were inconclusive in determining a link, as were those conducted using an ELISA developed to the adjacent C-terminal portion of the precursor peptide. Consequently, extraction and purification ofNKB from the human placenta was undertaken, with separation on the basis of size and hydrophobicity. A commercial one-site competitive NKB radioimmunoassay was used at each stage to identify immunoreactive fractions and matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry (MS) ,was performed on the purified samples. These experiments indicated that NKB extracted from the placenta had a higher molecular weight than NKB reported from the brain. The mass difference indicated one of two potential posttranslational modifications in the placenta of either a di-methylation or phosphocholine (PC) modification. These hypotheses were investigated through the development of specific two-site ELISAs. Putative processing mechanlsms were also explored through synthetic peptide digests with the enzyme carboxypeptidase B. Analysis of the digests via thin layer chromatography and MS proved that the dimethylation hampered exopeptidase processing. Quantitative PCR for the closely-related membrane-bound enzyme carboxypeptidase M, showed highest expression in the placenta. Synthetic NKB peptides carrying a dirnethylation however, had no biological activity either in in vitro ligand binding studies at the NK3 recepto~ or in in vivo models involving both rats and mice. Larger scale placental purification disproved the dimethylation theory and provided evidence for the PC modification thus far unprecedented in mammals, where pilot assays detected signals in both placental extracts and plasma samples. Collectively, these results provide evidence for the existence ofa unique placental NKB peptide, the structure of which has yet to be fully elucidated, as does the exact nature of the involvement ofthis peptide in PE.
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Munro, Fiona E. "The role of NK1 and NK2 neurokinin receptors in the acute and sustained nociceptive activation of dorsal horn neurons." Thesis, University of Edinburgh, 1997. http://hdl.handle.net/1842/29900.
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The tachykinins substance P (SP) and neurokinin A (NKA) can be released from fine somatosensory afferents into the spinal cord by noxious cutaneous stimuli. This study assessed the role of their respective NK1 and NK2 receptors and some putative intracellular mediators in both acute and sustained nociceptive inputs to dorsal horn neurons: (a) In anaesthetised rats, extracellular recordings were made from laminae III-V multireceptive neurons. The ionophoretic administration of NK1 antagonists L-668,169, GR 82334 and [D-Pro4,D-Trp7,9,10Phe11]substance P-(4-11) failed to influence neuronal responses to noxious pinch or heat, but often enhanced responses to innocuous brush, whilst the NK2 antagonist L-659,874 inhibited responses to noxious heat, but not pinch or brush. Selective NK1 and NK2 receptor agonists, [N-acetyl-Arg6,Sar9,Met(O2)11]SP6-11 and GR 64349 respectively, both excited dorsal horn neurons. The contribution of NK1 and NK2 receptors to sustained neuronal activity induced by peripheral application of the C-fibre selective algogen mustard oil was then investigated. (b) Evidence for a role of protein kinase C (PKC) in mediating sustained nociceptive responses of rat horn neurons was provided by the blockade of mustard oil-, but not brush-evoked neuronal activation by the PKC inhibitors GF 109203X and chelerythrine and by SR 48968-sensitive subcellular translocation of [3H]phorbol 12,13-dibutyrate binding sites ipsilateral to mustard oil stimulation. (c) In situ hybridisation histochemistry (ISSH) demonstrated that expression of c-fos mRNA, induced in the superficial dorsal horn by peripheral application of mustard oil was inhibited by systemic administration of both RP 67580 and SR 48968. (d) The effects of intrathecally-applied NK1 and NK2 antagonists were assessed on thermally-evoked tail-flick and paw-flick behavioural responses. GR 82334 and L-659,874 had no effect alone, but in combination inhibited paw-flick. After inflammation induced by intraplantar injection of carrageenan, each was effective individually. These results provide evidence that spinal NK2 receptors are involved in mediating both acute and sustained nociceptive transmission, probably acting through phosphoinositide hydrolysis and stimulation of protein kinase C(PKC). However, evidence for NK1 receptor involvement was only obtained in sustained or inflammatory models of nociceptive transmission in the spinal dorsal horn.
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Fourie, Christle. "Effects of clomiphene citrate on the expression of kisspeptin dynorphin A and neurokinin B in female Sprague-Dawley rats." Diss., University of Pretoria, 2016. http://hdl.handle.net/2263/61684.
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Clomiphene citrate (CC) is the leading treatment for women with anovulatory infertility. The precise mechanism of action of the drug on the hypothalamic-pituitary-gonadal (HPG) axis has yet to be determined. Neurons expressing kisspeptin, neurokinin B (NKB) and dynorphin A (Dyn), collectively called KNDy neurons, in the arcuate nucleus (ARC) of the hypothalamus have been shown to play an integral role in the estradiol (E2) feedback pathways of the reproductive system in mammals. KNDy neurons are found in the ARC and the anteroventral periventricular nucleus (AVPV) in humans but have been predominantly reported to not express NKB and Dyn in rodents. The axons of these neurons project to the medial eminence (ME) in the region where the gonadotropin-releasing hormone (GnRH) terminals and fibres are located. It was hypothesised that CC upregulates the gene expression of kisspeptin and neurokinin B while down-regulating the gene expression of dynorphin A which results in a leutenizing hormone surge and an increase in oestradiol which causes ovulation.
This was a randomized experiment which included 18 female Sprague-Dawley rats in which the aim was to analyse the expression of kisspeptin, NKB and Dyn in the ARC and the AVPV as well as blood plasma levels of oestradiol and leutinizing hormone (LH) in female rats after CC administration. Six of the rats constituted the control group that received a vehicle solution. The second group of 6 rats received the intervention in the form of CC and the third group of six rats received CC as well as p234-penetratin, a kisspeptin antagonist (KpA). The mRNA expression of the KNDy genes were analysed using real-time quantitative polymerase chain reaction (qPCR) and the plasma levels of E2 and LH were analysed by enzyme-linked immunosorbant assays (ELISA).
ELISA results show that the E2 concentration in the group that received CC plus KpA was found to be marginally lower than that of the control group but there was no significant difference between the E2 concentrations of the control group and the group that received only CC. The LH concentration in the group that received CC plus KpA was significantly higher than both other groups but again, there was no significant difference between the LH concentration control group and the group that only received CC. The qPCR showed that in the AVPV, the kisspeptin expression of the CC group and the CC plus KpA groups are marginally higher than that of the control group. Conversely, the Dyn expression of the CC group and the CC plus KpA groups are marginally lower than that of the control group in the AVPV. There were no significant differences in NKB expression across the three groups. In the ARC, there were no significant differences in kisspeptin or Dyn expression across the groups. The NKB expression of the CC group was marginally lower than that of the control and there was no significant difference between the CC plus KpA group and the control group. In summary, CC appears to have a marginal effect on the kisspeptin and Dyn mRNA via the positive feedback systems in the rat AVPV as well as a significant decrease of NKB mRNA via the negative feedback systems in the ARC. To increase the validity of similar future studies, higher sample sizes, different drug administration doses, possibly more precise surgical techniques and more accurate age determination methods or ovariectomised rats could be used.Dissertation (MSc)--University of Pretoria, 2016.
Physiology
MSc
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FORADORI, CHAD D. "THE ROLE OF ENDOGENOUS OPIOID PEPTIDES IN THE OVINE ESTROUS CYCLE." University of Cincinnati / OhioLINK, 2003. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1061305407.
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Young, Marie R. "The role of metabotropic glutamate and neurokinin receptors in mediating sustained nociceptive inputs to the spinal cord of the rat." Thesis, University of Edinburgh, 1995. http://hdl.handle.net/1842/30962.
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Glutamate, substance P (SP) and neurokinin A (NKA) are all found in fine primary afferent fibres and can be released upon noxious stimulation of the corresponding cutaneous receptive field. The possibility of a role in nociception for the metabotropic class of glutamate receptors (mGluRs) as well as those at which SP and NKA preferentially act (NK1 and NK2), was investigated in the present study. Since protein kinase C (PKC) has been shown to be important in the mediation of noxious, but not non-nociceptive inputs, the potential role of this and several other signal transduction pathways in sensory inputs was assessed here, especially in the context of actions via mGluRs. (a) Extracellular recordings were made from single dorsal horn neurons (laminae III-V) in the spinal cords of chloralose/urethane anaesthetised rats. Activity evoked by innocuous brushing of the cutaneous receptive field was not reduced by ionophoresis of mGluR antagonists L-1-amino-3-phosphopropionic acid (AP3), (R,S)- or (S)-4-carboxy-3-hydroxyphenylglycine (CHPG). This investigation demonstrates a role for spinal cord mGluRs in the transmission of sustained nociception, possibly mediated by PKC, CamKII and PLA2. NK2 receptors appear to have a selective role in thermal inputs to the spinal cord, whereas this study provided no evidence for an overt role of NK1 receptors in the nociceptive models assessed. It is possible that NK1 receptors play a greater role in more prolonged or severe nociceptive inputs. The present data suggest however that not only NK2, but also NK1 receptors exhibit a functional interaction with the influence of mGluRs on nociceptive thresholds.
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Naim, Magda Mohamed. "Primary afferent input to neurons in laminae III and IV of the rat spinal cord which possess the neurokinin-1 (NK-1) receptor." Thesis, University of Glasgow, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.298697.
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Gillespie, Earl. "Colonic epithelial genes in the transition from chronic inflammation to carcinoma in human colitis-associated cancer: focus on the truncated neurokinin-1 receptor." Thesis, Boston University, 2013. https://hdl.handle.net/2144/12761.
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Thesis (Ph.D.)--Boston University
PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.Patients with chronic ulcerative colitis (UC) are at high risk for developing colorectal cancer. Since there is a need for early detection and targeted therapeutics to treat this disease, the aim of this study was to identify changes in expression of pro-inflammatory and oncogenic genes and proteins in colonic tissue from patients with UC who developed colon cancer. Using laser capture microscopy, epithelia were microdissected from archival formalin-fixed paraffin-embedded colonic tissue that showed histologic evidence of carcinoma (CA), high-grade dysplasia (HGD), and non-neoplastic areas with evidence of prior inflammation (quiescent colitis, QC). mRNA was extracted from the dissected tissue and PCR array analysis was performed. Three proteins, plasminogen activator inhibitor-1 (PAI-1), the truncated neurokinin-1 receptor (tr-NK-1R) and its full-length isoform (fl-NK-1R), were quantitated using immunofluorescence. The biological properties of the tr-NK-1R and fl-NK-1R were further explored through stable transfection in HEK293 cells. mRNA expression of the tr-NK-1R is increased 14-fold (n=5, p=0.02) in carcinoma compared to HGD, while the fl-NK-1R transcript shows no significant differences amongst groups. mRNA expression of PAI-1 is increased 6-fold (n=5, p=0.02) in carcinoma compared to QC. Total NK-1R protein is increased by 40% (n=1 0, p=0.02) in HGD and by 80% (n=9, p=0.0007) in carcinoma compared to QC (n=11). There is no significant change in fl-NK-1R protein among groups, permitting the conclusion that the increase in total NK-1R protein in HGD and carcinoma is attributable to an increase in tr-NK-1R. PAI-1 protein is increased by 50% (n=1 0, p<0.001) in HGD and by 60% (n=9, p<0.001) in carcinoma compared to QC (n=11). HEK293 cells transfected with tr-NK-1R proliferate more rapidly. Tr-NK-1R transfectants also produce a greatly increased level of heat shock protein 70, an oncogenic protein that may participate in their proliferation. These data suggest there may be a functional role for PAI-1 and tr-NK-1R, perhaps through the upregulation of heat shock protein 70, in malignant transformation in colitis-associated cancer. The two proteins could prove useful as diagnostic markers to identify patients at risk for neoplasia and may serve as useful therapeutic targets in the treatment of colitis-associated cancer.
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Andersson, Gustav. "Influences of paratendinous innervation and non-neuronal substance P in tendinopathy : studies on human tendon tissue and an experimental model of Achilles tendinopathy." Doctoral thesis, Umeå universitet, Anatomi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-35917.
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Pain of the musculoskeletal system is one of the most common reasons for people seeking medical attention, and is also one of the major factors that prevent patients from working. Chronic tendon pain, tendinopathy, affects millions of workers world-wide, and the Achilles tendon is an important structure often afflicted by this condition. The pathogenesis of tendinopathy is poorly understood, but it is thought to be of multifactoral aetiology. It is known that tendon pain is often accompanied not only by impaired function but also by structural tissue changes, like vascular proliferation, irregular collagen organisation, and hypercellularity, whereby the condition is called tendinosis. In light of the poor knowledge of tendinosis pathophysiology and recent findings of a non-neuronal signalling system in tendon tissue, the contributory role of neuropeptides such as substance P (SP) has gained increased interest. SP, known for afferent pain signalling in the nervous system, also has multiple efferent functions and has been described to be expressed by non-neuronal cells. As pain is the most prominent symptom of tendinopathy, the focus of the studies in this thesis was the innervation patterns of the tissue ventral to the Achilles tendon (i.e. the tissue targeted in many contemporary treatment methods) as well as the distribution of SP and its preferred receptor, the neurokinin-1 receptor (NK-1R), in the tendon tissue itself. It was hereby hypothesised that the source of SP affecting the Achilles tendon might be the main cells of the tendon tissue (the tenocytes) as well as paratendinous nerves, and that SP might be involved in tendinosis- development. The studies were conducted, via morphological staining methods including immunohistochemistry and in situ hybridisation, on tendon biopsies from patients suffering from Achilles tendinosis and on those from healthy volunteers. The hypothesis of the thesis was furthermore tested using an experimental animal model (rabbit) of Achilles tendinopathy, which was first validated. The model was based on a previously established overuse protocol of repetitive exercise. In the human biopsies of the tissue ventral to the Achilles tendon, there was a marked occurrence of sympathetic innervation, but also sensory, SP-containing, nerve fibres. NK-1R was expressed on blood vessels and nerve fascicles of the paratendinous tissue, but also on the tenocytes of the tendon tissue proper itself, and notably more so in patients suffering from tendinosis. Furthermore, the human tenocytes displayed not only NK-1R mRNA but also mRNA for SP. The animal model was shown to produce objectively verified tendinosis-like changes, such as hypercellularity and increased vascularity, in the rabbit Achilles tendons, after a minimum of three weeks of the exercise protocol. The contralateral leg of the animals in the model was found to be an unreliable control, as bilateral changes occured. The model furthermore demonstrated that exogenously administered SP triggers an inflammatory response in the paratendinous tissue and accelerates the intratendinous tendinosis-like changes such that they now occur after only one week of the protocol. Injections of saline as a control showed similar results as SP concerning hypercellularity, but did not lead to vascular changes or pronounced paratendinous inflammation. In summary, this thesis concludes that interactions between the peripheral sympathetic and sensory nervous systems may occur in Achilles tendinosis at the level of the ventral paratendinous tissue, a region thought to be of great importance in chronic tendon pain since many successful treatments are directed toward it. Furthermore, the distribution of NK-1R:s in the Achilles tendon described in these studies gives a basis for SP, whether produced by nerves mainly outside the tendon or by tenocytes within the tendon, to affect blood vessels, nerve structures, and/or tendon cells, especially in tendinosis patients. In light of this and of previously known SP-effects, such as stimulation of angiogenesis, pain signalling, and cell proliferation, the proposed involvement of SP in tendinosis development seems likely. Indeed, the animal model of Achilles tendon overuse confirms that SP does induce vascular proliferation and hypercellularity in tendon tissue, thus strengthening theories of SP playing a role in tendinosis pathology.
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Porter, A. J. "The role of the neurokinin-1 receptor in behaviour and cognition : an interaction with the Brain Renin Angiotensin System and its implications for Attention Deficit Hyperactivity Disorder." Thesis, University College London (University of London), 2015. http://discovery.ucl.ac.uk/1466740/.
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Mice lacking functional neurokinin-1 receptors (NK1R-/-) display several behavioural abnormalities that resemble Attention Deficit Hyperactivity Disorder (ADHD), including locomotor hyperactivity (which is alleviated by ADHD medication), impulsivity and inattentiveness. These findings prompted the proposal that NK1R-/- mice offer a novel murine model of ADHD. The first aim of this thesis was to investigate the influence of non-genetic (epigenetic/environmental) factors on the behaviour of NK1R-/- mice by comparing animals from two different breeding protocols. These studies revealed that certain elements of their impulsivity are influenced by non-genetic factors, but other behaviours are not. The second aim was to compare the behaviour of male and female animals, to determine whether there are sex differences in their behaviour. This study revealed that the hyperactivity of NK1R-/- mice is not evident in females, echoing typical sex differences in ADHD patients. Following this, the behavioural effects of several drugs that target the brain renin angiotensin system (BRAS) are reported. This was prompted by preliminary evidence that the BRAS and NK1R interact in the regulation of locomotor activity. The angiotensin converting enzyme (ACE) inhibitor, captopril, abolished the hyperactivity and impulsivity of (male) NK1R-/- mice, suggesting that ACE could provide a novel therapeutic target for the treatment of ADHD. By contrast, angiotensin receptor antagonism either exacerbated or had no effect on these behaviours, suggesting that captopril's therapeutic effect is not due to a reduction in angiotensin II. Finally, the performance of NK1R-/- mice in the 5-Choice Continuous Performance Test was investigated. NK1R-/- mice displayed performance deficits only during certain phases of training, suggesting that detection of the impulsive or inattentive phenotype might require unpredictable test parameters. Overall, these studies further validate/phenotype the NK1R-/- mouse model of ADHD, and point to an interaction between the BRAS and NK1R in the regulation of ADHD-related behaviours, which warrants further investigation.
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Tusset, Cíntia. "Pesquisa de mutações na neurocinina B e no seu receptor em pacientes com distúrbios puberais centrais idiopáticos." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/5/5135/tde-03092012-090655/.
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Mutações inativadoras nos genes TAC3 e TACR3, os quais codificam a neurocinina B (NKB) e o seu receptor NK3R, respectivamente, foram descritas em pacientes com hipogonadismo hipogonadotrófico isolado (HHI) normósmico. A partir desse achado, hipotetizamos que mutações ativadoras na NKB e/ou NK3R resultariam na secreção prematura de GnRH e, consequentemente, no desenvolvimento de puberdade precoce dependente de gonadotrofinas (PPDG). Nesse estudo, investigamos a presença de mutações ativadoras e/ou polimorfismos nos genes TAC3 e TACR3 em pacientes com PPDG, bem como mutações inativadoras e/ou polimorfismos nesses genes em pacientes com retardo constitucional do crescimento e desenvolvimento (RCCD), e HHI normósmico. Duzentos e trinta e sete pacientes com distúrbios puberais centrais idiopáticos foram selecionados, sendo 114 com PPDG, 50 com RCCD, e 73 com HHI normósmico. Um grupo de 150 indivíduos que apresentaram desenvolvimento puberal normal foi utilizado como controle. As regiões codificadoras dos genes TAC3 e TACR3 foram amplificadas pela reação em cadeia da polimerase, seguido de purificação enzimática e seqüenciamento automático direto. Análises in silico e in vitro foram realizadas. Um nova variante foi identificada no gene TAC3, p.A63P, em uma paciente do sexo feminino com PPDG, a qual desenvolveu puberdade aos sete anos de idade. Essa variante (p.A63P) está localizada na proneurocinina B, e análises in silico sugeriram que ela não altera sítios constitutivos de splicing e é benigna para a estrutura da proteína. A análise de segregação familiar mostrou que a mãe da paciente, a qual apresentou um desenvolvimento puberal normal, também apresentava a alteração p.A63P em heterozigose, sugerindo que essa variante não desempenha um papel direto no fenótipo de PPDG. Uma nova variante em heterozigose no gene TACR3, p.A449S, foi identificada em uma paciente do sexo feminino com RCCD, que teve início puberal aos treze anos de idade. A análise do grau de conservação da alanina na posição 449 mostrou que esse aminoácido não é conservado entre as diferentes espécies, e análises in silico sugeriram que essa variante não altera os sítios constitutivos de splicing, e é benigna para a estrutura do NK3R. Três novas variantes no NK3R foram identificadas, p.G18D, p.L58L (c.172C>T) e p.W275*, em três pacientes do sexo masculino não relacionados com HHI normósmico. As variantes p.G18D e p.L58L foram identificadas em heterozigose, enquanto que a variante p.W275* foi identificada em heterozigose associada a variante silenciosa p.L58L (c.172C>T), e em homozigose em outro paciente. Análises in silico sugeriram que a variante p.G18D poderia afetar a funcionalidade do NK3R. Estudos in vitro dessa nova variante foram realizados, e mostraram que a mesma não altera a função do NK3R, visto que o aumento na produção de fosfatidil inositol não diferiu significativamente entre o receptor mutado e selvagem. Todas as novas variantes descritas nos genes TAC3 e TACR3 não foram identificadas em 300 alelos controles. Em conclusão, nosso trabalho identificou novas variantes nos genes TAC3 e TACR3 em pacientes brasileiros com distúrbios puberais centrais idiopáticos, e confirmou o envolvimento do complexo NKB/NK3R na etiologia do HHI normósmicoInactivating mutations of the TAC3 and TACR3 genes, which encode the neurokinin B (NKB) and its receptor, NK3R, respectively, were described in patients with normosmic isolated hypogonadotropic hypogonadism (IHH). Based on these observations, we hypothesized that gain-of-function mutations in the NKB and/or NK3R might be associated with premature activation of GnRH release, leading to gonadotropin-dependent precocious puberty (GDPP). In this study, we investigated the presence of activating mutations and/or polymorphisms in the TAC3 and TACR3 genes in patients with GDPP, and inactivating mutations and/or polymorphisms in these genes in patients with constitutional delay of growth and puberty (CDGP) and normosmic IHH. It was selected 237 patients with idiopathic central pubertal disorders: 114 with GDPP, 50 with CDGP, and 73 with normosmic IHH. Indeed, a group 150 individuals who had puberty at adequate age was used as controls. The coding regions of TAC3 and TACR3 genes were amplified by polymerase chain reaction followed by enzymatic purification and direct automatic sequencing. In silico and in vitro analyses were performed. A new heterozygous variant in the TAC3 gene, p.A63P, was identified in a Brazilian girl with GDPP who had puberty onset at seven years of age. The p.A63P variant was located in the proneurokinin B and in silico analysis suggested that this variant does not alter constitutive splice sites, and it was benign to the protein. The segregation analysis revealed that her mother was heterozygous for the p.A63P variant (who had a normal pubertal development), suggesting that this variant does not play a role in the GDPP phenotype. It was identified a new heterozygous variant, p.A449S, in the TACR3 gene in a Brazilian girl with CDGP, who had puberty onset at thirteen years of age. Conservation degree analysis of alanine at position 449 showed that this amino acid is not a conserved residue among different species. In silico analyses suggested that this new variant does not alter splice sites or affects the structure of NK3R. Indeed, it was identified three new distinct variants in the TACR3 gene, p.G18D, p.L58L (c.172C>T) and p.W275*, in three unrelated males with normosmic IHH. Both p.G18D and p.L58L (c.172C>T) were identified in heterozygous state, and the p.W275* variant was identified in two of these males, since one in homozygous and in another in heterozygous state in association with the silent variant p.L58L (c.172C>T). In silico analyses suggested that p.G18D might be damaging to the NK3R. In vitro studies of this variant (p.G18D) showed that the amount of inositol phosphate (IP) was not significantly different in cells transfected with the p.G18D mutant receptor than in cells transfected with the wild type receptor, indicating that this variant did not alter the function of the neurokinin B receptor. All new variants identified in the TAC3 and TACR3 genes were absent in 300 control alleles. In conclusion, we identified new variants in the TAC3 and TACR3 genes in Brazilian patients with idiopathic central pubertal disorders. We confirm the key role of the NKB/NK3R complex in the etiology of normosmic IHH
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Min, Byoung Joon. "PART I. DESIGN AND SYNTHESIS OF BICYCLIC INTERNAL BETA-TURN MIMETICS AND THEIR INCORPORATION INTO BIOLOGICALLY ACTIVE LIGANDS; PART II. SYNTHESIS OF CYCLIC PEPTIDES BY RING." Diss., The University of Arizona, 2010. http://hdl.handle.net/10150/194081.
Full textAbstract:
beta-Turns in many biologically active peptides are important secondary structural elements which are critical for their biological activities. Hence, it is not surprising that beta-turn based pharmacophore design including beta-turn mimetics has become a central topic in medicinal chemistry in addition to alpha-helix or helical peptides. One of the advantages of such beta-turn mimetics is that they can better control torsion angles of the backbone of peptides and to some degree dihedral angles chi (X). These beta-turn mimicking scaffolds are designed to have a higher avidity for the acceptor by overcoming what otherwise is the inherent entropic cost paid for beta-turn formation upon binding to the acceptor. Among different synthetic strategies to bicyclic structures as beta-turn mimetics, consecutive formation of bicyclic structures using tandem acid-catalyzed N-acyliminium ion cyclization is attractive since this methodology was well established in the synthesis of natural product alkaloids. 1,3,6,8-Substituted tetrahydro-2H-pyrazino[1,2-a]pyrimidine-4,7-diones were designed and synthesized as internal beta-turn mimetics through an acid-catalyzed tandem acyliminium ion cyclization. Its development and synthesis are decribed in Chapter 2 to Chapter 4. Its application toward the development and synthesis of a small molecule ligand for melanocortin receptors is described in Chapter 5. In addition, the development of peptidomimetics for opioid receptors is explained in Chapter 6. On the other hand, a dicarba analogue having opioid receptor agonist, and dicarba analogues for MCRs were synthesized through solid phase synthesis including a ring closing metathesis reaction using Grubbs' catalyst (I) in Chapter 8.