3,967 research outputs found

    On the G-protein-coupled receptor heteromers and their allosteric receptor-receptor interactions in the central nervous system: focus on their role in pain modulation

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    The modulatory role of allosteric receptor-receptor interactions in the pain pathways of the Central Nervous System and the peripheral nociceptors has become of increasing interest. As integrators of nociceptive and antinociceptive wiring and volume transmission signals, with a major role for the opioid receptor heteromers, they likely have an important role in the pain circuits and may be involved in acupuncture. The delta opioid receptor (DOR) exerts an antagonistic allosteric influence on the mu opioid receptor (MOR) function in a MOR-DOR heteromer. This heteromer contributes to morphine-induced tolerance and dependence, since it becomes abundant and develops a reduced G-protein-coupling with reduced signaling mainly operating via beta-arrestin 2 upon chronic morphine treatment. A DOR antagonist causes a return of the Gi/o binding and coupling to the heteromer and the biological actions of morphine. The gender- and ovarian steroid-dependent recruitment of spinal cord MOR/kappa opioid receptor (KOR) heterodimers enhances antinociceptive functions and if impaired could contribute to chronic pain states in women. MOR1D heterodimerizes with gastrin-releasing peptide receptor (GRPR) in the spinal cord, mediating morphine induced itch. Other mechanism for the antinociceptive actions of acupuncture along meridians may be that it enhances the cross-desensitization of the TRPA1 (chemical nociceptor)-TRPV1 (capsaicin receptor) heteromeric channel complexes within the nociceptor terminals located along these meridians. Selective ionotropic cannabinoids may also produce cross-desensitization of the TRPA1-TRPV1 heteromeric nociceptor channels by being negative allosteric modulators of these channels leading to antinociception and antihyperalgesia

    Distinct roles of NMB and GRP in itch transmission

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    A key question in our understanding of itch coding mechanisms is whether itch is relayed by dedicated molecular and neuronal pathways. Previous studies suggested that gastrin-releasing peptide (GRP) is an itch-specific neurotransmitter. Neuromedin B (NMB) is a mammalian member of the bombesin family of peptides closely related to GRP, but its role in itch is unclear. Here, we show that itch deficits in mice lacking NMB or GRP are non-redundant and Nmb/Grp double KO (DKO) mice displayed additive deficits. Furthermore, both Nmb/Grp and Nmbr/Grpr DKO mice responded normally to a wide array of noxious stimuli. Ablation of NMBR neurons partially attenuated peripherally induced itch without compromising nociceptive processing. Importantly, electrophysiological studies suggested that GRPR neurons receive glutamatergic input from NMBR neurons. Thus, we propose that NMB and GRP may transmit discrete itch information and NMBR neurons are an integral part of neural circuits for itch in the spinal cord

    Circuit dissection of the role of somatostatin in itch and pain

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    Stimuli that elicit itch are detected by sensory neurons that innervate the skin. This information is processed by the spinal cord; however, the way in which this occurs is still poorly understood. Here we investigated the neuronal pathways for itch neurotransmission, particularly the contribution of the neuropeptide somatostatin. We find that in the periphery, somatostatin is exclusively expressed in Nppb+ neurons, and we demonstrate that Nppb+somatostatin+ cells function as pruriceptors. Employing chemogenetics, pharmacology and cell-specific ablation methods, we demonstrate that somatostatin potentiates itch by inhibiting inhibitory dynorphin neurons, which results in disinhibition of GRPR+ neurons. Furthermore, elimination of somatostatin from primary afferents and/or from spinal interneurons demonstrates differential involvement of the peptide released from these sources in itch and pain. Our results define the neural circuit underlying somatostatin-induced itch and characterize a contrasting antinociceptive role for the peptide

    Expression of gastrin-releasing peptide by excitatory interneurons in the mouse superficial dorsal horn

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    Background: Gastrin-releasing peptide (GRP) and its receptor have been shown to play an important role in the sensation of itch. However, although GRP immunoreactivity has been detected in the spinal dorsal horn, there is debate about whether this originates from primary afferents or local excitatory interneurons. We therefore examined the relation of GRP immunoreactivity to that seen with antibodies that label primary afferent or excitatory interneuron terminals. We tested the specificity of the GRP antibody by preincubating with peptides with which it could potentially cross-react. We also examined tissue from a mouse line in which enhanced green fluorescent protein (EGFP) is expressed under control of the GRP promoter.<p></p> Results: GRP immunoreactivity was seen in both primary afferent and non-primary glutamatergic axon terminals in the superficial dorsal horn. However, immunostaining was blocked by pre-incubation of the antibody with substance P, which is present at high levels in many nociceptive primary afferents. EGFP+ cells in the GRP-EGFP mouse did not express Pax2, and their axons contained the vesicular glutamate transporter 2 (VGLUT2), indicating that they are excitatory interneurons. In most cases, their axons were also GRP-immunoreactive. Multiple-labelling immunocytochemical studies indicated that these cells did not express either of the preprotachykinin peptides, and that they generally lacked protein kinase CÎł, which is expressed by a subset of the excitatory interneurons in this region.<p></p> Conclusions: These results show that GRP is expressed by a distinct population of excitatory interneurons in laminae I-II that are likely to be involved in the itch pathway. They also suggest that the GRP immunoreactivity seen in primary afferents in previous studies may have resulted from cross-reaction of the GRP antibody with substance P or the closely related peptide neurokinin A

    Applications of siRNA for Cancer Gene Therapy

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    Gene therapy is a potent and versatile nano-medicine strategy in the treatment of cancer. Of the many tools currently used in this application, short-interfering RNA (siRNA) are among the most commonly employed due to their ability to silence oncogenic mRNA with high precision through the RNA interference (RNAi) pathway potentially leading to cancer cell death. Our work revolves around silencing the Glucose-Regulated Proteins (GRPs) whose expressions are upregulated in cancerous tissues and are implicated in the proliferative, pro-survival, and anti-apoptotic pathways that govern tumor biology. Here we present a variety of applications to improve the potency and functionality of GRP-targeting siRNAs while investigating the role of these oncoproteins in cancer cell cytotoxicity and adhesion. We’ve generated branched RNA templates that have the ability to self-assemble into higher order siRNA nanostructures and are capable of delivering multiple siRNAs that can synergistically silence the GRPs in endometrial, breast, and cervical cancer. From the branched RNA templates, second generation bioconjugates were developed to include fluorescently labeled and fatty acid conjugated siRNAs in an effort to expand their functionality into theranostic and self-delivery applications. Using our silencing strategy, we’ve also revealed a novel role of the master GRP regulator, GRP78, in modulating the expression of N-cadherin, a mesenchymal marker pivotal in the progression of metastatic tumor cell adhesion to the bone microenvironment. Silencing of GRP78 ultimately led to a decrease in prostate cancer adhesion in a model bone co-culture system. Moreover, our latest work highlights a method for developing cancer-targeting and cell penetrating peptides for the targeted delivery of siRNA to prostate cancer cells. Together, these studies have introduced new technologies that can further enhance the potency and applicability of siRNAs in cancer gene therapy

    Engineered antibody and neuropeptide mediated radionuclide targeting in prostate cancer

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    PhDProstate cancer (PC) is the most common cancer type in men in the western world and to date no definitive stratergy to image PC is avaliable. This thesis explores the possibility of using Prostate Specific Membrane Antigen (PSMA) and Gastrin Releasing Peptide Receptor (GRP-R) as biomarkers for the targeting and imaging of PC. The development of an imaging radiopharmaceutical to image all stages of PC growth would improve diagnosis, staging and personalised treatment, as present imaging modalities for PC rely largely on anatomical changes to allow visualisation and have limited sensitivity for imaging metastatic spread of the disease. PSMA was selected due to its up-regulation in advanced carcinoma and metastatic disease and GRP-R due to its high levels of expression in the early stages of PC. The hypothesis is that PC can be imaged by a suitably designed radioligand directed against an appropriate molecular target, such as PSMA and GRP-R. Both of these targets were believed to be appropriate as both are present preferntially in prostate tissue and they both internalise when bound by their ligand. To target PSMA, phage libraries were screened for scFv against both cell-expressed PSMA and recombinant PSMA and diabodies were also generated from high binding clones. Several promising candidates were produced which selectively bound to LNCaP cells and PSMA protein in both FACS and ELISA. Diabodies showed improved binding over corresponding scFv’s. In vivo analysis of tumour-bearing mice failed to reveal tumour uptake of either the scFv or the diabody. In vitro analysis suggested that the affinity of the antibody fragments were not sufficiently high. [99mTc]-Demobesin 4 (DB 4), a radiolabelled GRP-R binding peptide was synthesised. Radioligand binding assays performed on a range of androgen-independent and androgen-dependent PC cell lines showed high GRP-R expression in the androgen dependent LNCaP line but also in the androgen-independent cell lines PC3 and DU145. GRP-R expression, measured by RT-PCR to determine the amount of GRP-R RNA, was similar to that seen using radioligand binding assays and similar patterns were observed in autoradiographic studies. In vivo studies on mice bearing the PC xenografts showed tumour uptake and localisation of [99mTc]-DB 4 within one hour. A limited correlation was observed between results obtained in vivo and in vitro. In conclusion, the results were partly consistant with the hypothesis, whereby initial aims for the PSMA project were successfully achieved with generation of scFv and diabodies that specifically bound, however they proved unsuitable as potential imaging agents, perhaps owing to low binding affinity. GRP-R was shown to be an effective candidate for radioimmaging PC which has the potential to descrininate [99mTc]-DB4 uptake between androgen-independent/dependent cells. Thus this radiopharmaceutical may prove a useful imaging agent for early prostate cancer but that further studies are required to assess its usefulness in the androgen-independent stages of the disease

    Preprotachykinin A (PPTA) is expressed by a distinct population of excitatory neurons in the mouse superficial spinal dorsal horn including cells that respond to noxious and pruritic stimuli

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    The superficial dorsal horn, which is the main target for nociceptive and pruritoceptive primary afferents, contains a high density of excitatory interneurons. Our understanding of their roles in somatosensory processing has been restricted by the difficulty of distinguishing functional populations among these cells. We recently defined three non-overlapping populations among the excitatory neurons, based on the expression of neurotensin, neurokinin B (NKB) and gastrin-releasing peptide (GRP). Here we identify and characterise another population: neurons that express the tachykinin peptide substance P. We show with immunocytochemistry that its precursor protein (preprotachykinin A, PPTA) can be detected in ~14% of lamina I-II neurons, and these are concentrated in the outer part of lamina II. Over 80% of the PPTA-positive cells lack the transcription factor Pax2 (which determines an inhibitory phenotype), and these account for ~15% of the excitatory neurons in this region. They are different from the neurotensin, NKB or GRP neurons, although many of them contain somatostatin, which is widely expressed among superficial dorsal horn excitatory interneurons. We show that many of these cells respond to noxious thermal and mechanical stimuli, and to intradermal injection of pruritogens. Finally, we demonstrate that these cells can also be identified in a knock-in Cre mouse line (Tac1Cre), although our findings suggest that there is an additional population of neurons that transiently express PPTA. This population of substance P-expressing excitatory neurons is likely to play an important role in transmission of signals that are perceived as pain and itch
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