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Blum NK, Schaffner A, Drube J, Nagel F, Reinscheid RK, Hoffmann C, Schulz S. Rapid elucidation of agonist-driven regulation of the neurokinin 1 receptor using a GPCR phosphorylation immunoassay. Eur J Pharmacol 2024; 973:176587. [PMID: 38642667 DOI: 10.1016/j.ejphar.2024.176587] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2024] [Revised: 04/09/2024] [Accepted: 04/11/2024] [Indexed: 04/22/2024]
Abstract
Agonist-induced phosphorylation is a crucial step in the activation/deactivation cycle of G protein-coupled receptors (GPCRs), but direct determination of individual phosphorylation events has remained a major challenge. We have recently developed a bead-based immunoassay for the quantitative assessment of agonist-induced GPCR phosphorylation that can be performed entirely in 96-well plates, thus eliminating the need for western blot analysis. In the present study, we adapted this assay to three novel phosphosite-specific antibodies directed against the neurokinin 1 (NK1) receptor, namely pS338/pT339-NK1, pT344/pS347-NK1, and pT356/pT357-NK1. We found that substance P (SP) stimulated concentration-dependent phosphorylation of all three sites, which could be completely blocked in the presence of the NK1 receptor antagonist aprepitant. The other two endogenous ligands of the tachykinin family, neurokinin A (NKA) and neurokinin B (NKB), were also able to induce NK1 receptor phosphorylation, but to a much lesser extent than substance P. Interestingly, substance P promoted phosphorylation of the two distal sites more efficiently than that of the proximal site. The proximal site was identified as a substrate for phosphorylation by protein kinase C. Analysis of GPCR kinase (GRK)-knockout cells revealed that phosphorylation was mediated by all four GRK isoforms to similar extents at the T344/S347 and the T356/T357 cluster. Knockout of all GRKs resulted in abolition of all phosphorylation signals highlighting the importance of these kinases in agonist-mediated receptor phosphorylation. Thus, the 7TM phosphorylation assay technology allows for rapid and detailed analyses of GPCR phosphorylation.
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Affiliation(s)
- Nina K Blum
- Institut für Pharmakologie und Toxikologie, Universitätsklinikum Jena, Friedrich-Schiller-Universität Jena, Drackendorfer Str. 1, D-07747, Jena, Germany
| | - Anne Schaffner
- Institut für Pharmakologie und Toxikologie, Universitätsklinikum Jena, Friedrich-Schiller-Universität Jena, Drackendorfer Str. 1, D-07747, Jena, Germany
| | - Julia Drube
- Institut für Molekulare Zellbiologie, CMB - Center for Molecular Biomedicine, Universitätsklinikum Jena, Friedrich-Schiller-Universität Jena, Hans-Knöll-Str. 2, D-07745, Jena, Germany
| | - Falko Nagel
- 7TM Antibodies GmbH, Hans-Knöll-Str. 6, D-07745, Jena, Germany
| | - Rainer K Reinscheid
- Institut für Pharmakologie und Toxikologie, Universitätsklinikum Jena, Friedrich-Schiller-Universität Jena, Drackendorfer Str. 1, D-07747, Jena, Germany
| | - Carsten Hoffmann
- Institut für Molekulare Zellbiologie, CMB - Center for Molecular Biomedicine, Universitätsklinikum Jena, Friedrich-Schiller-Universität Jena, Hans-Knöll-Str. 2, D-07745, Jena, Germany
| | - Stefan Schulz
- Institut für Pharmakologie und Toxikologie, Universitätsklinikum Jena, Friedrich-Schiller-Universität Jena, Drackendorfer Str. 1, D-07747, Jena, Germany; 7TM Antibodies GmbH, Hans-Knöll-Str. 6, D-07745, Jena, Germany.
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2
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Chen J, Ye P, Gu R, Zhu H, He W, Mu X, Wu X, Pang H, Han F, Nie X. Neuropeptide substance P: A promising regulator of wound healing in diabetic foot ulcers. Biochem Pharmacol 2023; 215:115736. [PMID: 37549795 DOI: 10.1016/j.bcp.2023.115736] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2023] [Revised: 08/01/2023] [Accepted: 08/02/2023] [Indexed: 08/09/2023]
Abstract
In the past, neuropeptide substance P (SP) was predominantly recognized as a neuroinflammatory factor, while its potent healing activity was overlooked. This paper aims to review the regulatory characteristics of neuropeptide SP in both normal and diabetic wound healing. SP actively in the regulation of wound healing-related cells directly and indirectly, exhibiting robust inflammatory properties, promoting cell proliferation and migration and restoring the activity and paracrine ability of skin cells under diabetic conditions. Furthermore, SP not only regulates healing-related cells but also orchestrates the immune environment, thereby presenting unique and promising application prospects in wound intervention. As new SP-based preparations are being explored, SP-related drugs are poised to become an effective therapeutic intervention for diabetic foot ulcers (DFU).
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Affiliation(s)
- Jitao Chen
- College of Pharmacy, Zunyi Medical University, Zunyi 563000, China; Key Lab of the Basic Pharmacology of the Ministry of Education & Joint International Research Laboratory of Ethnomedicine of Chinese Ministry of Education, Zunyi Medical University, Zunyi 563000, China
| | - Penghui Ye
- College of Pharmacy, Zunyi Medical University, Zunyi 563000, China; Key Lab of the Basic Pharmacology of the Ministry of Education & Joint International Research Laboratory of Ethnomedicine of Chinese Ministry of Education, Zunyi Medical University, Zunyi 563000, China
| | - Rifang Gu
- University Medical Office, Zunyi Medical University, Zunyi 563000, China
| | - Huan Zhu
- College of Pharmacy, Zunyi Medical University, Zunyi 563000, China; Key Lab of the Basic Pharmacology of the Ministry of Education & Joint International Research Laboratory of Ethnomedicine of Chinese Ministry of Education, Zunyi Medical University, Zunyi 563000, China
| | - Wenjie He
- College of Pharmacy, Zunyi Medical University, Zunyi 563000, China; Key Lab of the Basic Pharmacology of the Ministry of Education & Joint International Research Laboratory of Ethnomedicine of Chinese Ministry of Education, Zunyi Medical University, Zunyi 563000, China
| | - Xingrui Mu
- College of Pharmacy, Zunyi Medical University, Zunyi 563000, China; Key Lab of the Basic Pharmacology of the Ministry of Education & Joint International Research Laboratory of Ethnomedicine of Chinese Ministry of Education, Zunyi Medical University, Zunyi 563000, China
| | - Xingqian Wu
- College of Pharmacy, Zunyi Medical University, Zunyi 563000, China; Key Lab of the Basic Pharmacology of the Ministry of Education & Joint International Research Laboratory of Ethnomedicine of Chinese Ministry of Education, Zunyi Medical University, Zunyi 563000, China
| | - Huiwen Pang
- Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072, Australia
| | - Felicity Han
- Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072, Australia
| | - Xuqiang Nie
- College of Pharmacy, Zunyi Medical University, Zunyi 563000, China; Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072, Australia; Key Lab of the Basic Pharmacology of the Ministry of Education & Joint International Research Laboratory of Ethnomedicine of Chinese Ministry of Education, Zunyi Medical University, Zunyi 563000, China.
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Safwat A, Helmy A, Gupta A. The Role of Substance P Within Traumatic Brain Injury and Implications for Therapy. J Neurotrauma 2023; 40:1567-1583. [PMID: 37132595 DOI: 10.1089/neu.2022.0510] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/04/2023] Open
Abstract
This review examines the role of the neuropeptide substance P within the neuroinflammation that follows traumatic brain injury. It examines it in reference to its preferential receptor, the neurokinin-1 receptor, and explores the evidence for antagonism of this receptor in traumatic brain injury with therapeutic intent. Expression of substance P increases following traumatic brain injury. Subsequent binding to the neurokinin-1 receptor results in neurogenic inflammation, a cause of deleterious secondary effects that include an increased intracranial pressure and poor clinical outcome. In several animal models of TBI, neurokinin-1 receptor antagonism has been shown to reduce brain edema and the resultant rise in intracranial pressure. A brief overview of the history of substance P is presented, alongside an exploration into the chemistry of the neuropeptide with a relevance to its functions within the central nervous system. This review summarizes the scientific and clinical rationale for substance P antagonism as a promising therapy for human TBI.
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Affiliation(s)
- Adam Safwat
- Division of Anaesthesia, Department of Medicine, University of Cambridge, Cambridge, United Kingdom
- Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom
| | - Adel Helmy
- Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom
| | - Arun Gupta
- Neurosciences Critical Care Unit, Addenbrooke's Hospital, Cambridge, United Kingdom
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4
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Zhu Z, Bhatia M. Inflammation and Organ Injury the Role of Substance P and Its Receptors. Int J Mol Sci 2023; 24:6140. [PMID: 37047113 PMCID: PMC10094202 DOI: 10.3390/ijms24076140] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2023] [Revised: 03/17/2023] [Accepted: 03/22/2023] [Indexed: 04/14/2023] Open
Abstract
Tightly controlled inflammation is an indispensable mechanism in the maintenance of cellular and organismal homeostasis in living organisms. However, aberrant inflammation is detrimental and has been suggested as a key contributor to organ injury with different etiologies. Substance P (SP) is a neuropeptide with a robust effect on inflammation. The proinflammatory effects of SP are achieved by activating its functional receptors, namely the neurokinin 1 receptor (NK1R) receptor and mas-related G protein-coupled receptors X member 2 (MRGPRX2) and its murine homolog MRGPRB2. Upon activation, the receptors further signal to several cellular signaling pathways involved in the onset, development, and progression of inflammation. Therefore, excessive SP-NK1R or SP-MRGPRX2/B2 signals have been implicated in the pathogenesis of inflammation-associated organ injury. In this review, we summarize our current knowledge of SP and its receptors and the emerging roles of the SP-NK1R system and the SP-MRGPRX2/B2 system in inflammation and injury in multiple organs resulting from different pathologies. We also briefly discuss the prospect of developing a therapeutic strategy for inflammatory organ injury by disrupting the proinflammatory actions of SP via pharmacological intervention.
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Affiliation(s)
| | - Madhav Bhatia
- Department of Pathology and Biomedical Science, University of Otago, Christchurch 8140, New Zealand
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Alexander DP, Nickman NA, Chhibber A, Stoddard GJ, Biskupiak JE, Munger MA. Angiotensin-converting enzyme inhibitors reduce community-acquired pneumonia hospitalization and mortality. Pharmacotherapy 2022; 42:890-897. [PMID: 36278479 DOI: 10.1002/phar.2739] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2022] [Revised: 10/14/2022] [Accepted: 10/14/2022] [Indexed: 11/13/2022]
Abstract
BACKGROUND Pneumonia is a global disorder and a common reason for prolonged hospitalization. Angiotensin-converting enzyme inhibitors (ACEi) have pleiotropic effects that support a role in modulating pneumonia, but results have been controversial. OBJECTIVES The present study was conducted to elucidate an ACEi-induced pneumonia benefit in at-risk neurologically impaired population and to determine whether a mortality benefit exists. METHODS A cohort study using a large health-system of 29,011 unique ACEi users and 1635 case patients 65 years of age or older without neurological disorders affecting swallowing who were admitted with community-acquired pneumonia hospitalization and followed up from January 1, 2015 to December 31, 2019 (5 years). The association between ACEi use and pneumonia hospitalization and mortality were determined after propensity score matching using Cox and logistic regression. RESULTS The experimental cohort was 74.9 ± 7.3 years and 51% were male. ACEi users had lower odds of acquiring pneumonia versus ACEi non-users (odds ratio) 0.72 [95% Confidence Interval (CI) 0.51 to 0.99]; p = 0.048. The risk of short-term mortality (<30 days) (HR) 0.42, p < 0.001 and long-term mortality (≥30 day) (HR) 0.83, p < 0.002 was significantly lower for ACEi users compared with the ACEi non-users. CONCLUSIONS ACEi use in patients at risk of pneumonia without neurological swallowing disorders is associated with reduction in hospitalization and lowering of short- and long-term mortality. Given the high incidence of morbidity and mortality associated with pneumonia, and the susceptibility in older populations with underlying cardiovascular or renal disease or social dependencies, our data support the prescribing of ACEi in these populations to reduce pneumonia hospitalization risk as well as short- and long-term mortality.
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Affiliation(s)
- Donald P Alexander
- Department of Pharmacotherapy, University of Utah, Salt Lake City, Utah, USA
| | - Nancy A Nickman
- Department of Pharmacotherapy, University of Utah, Salt Lake City, Utah, USA
| | - Anindit Chhibber
- Department of Pharmacotherapy, University of Utah, Salt Lake City, Utah, USA
| | - Gregory J Stoddard
- Department of Internal Medicine, University of Utah, Salt Lake City, Utah, USA
| | - Joseph E Biskupiak
- Department of Pharmacotherapy, University of Utah, Salt Lake City, Utah, USA
| | - Mark A Munger
- Department of Pharmacotherapy, University of Utah, Salt Lake City, Utah, USA.,Department of Internal Medicine, University of Utah, Salt Lake City, Utah, USA
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Puri S, Kenyon BM, Hamrah P. Immunomodulatory Role of Neuropeptides in the Cornea. Biomedicines 2022; 10:1985. [PMID: 36009532 PMCID: PMC9406019 DOI: 10.3390/biomedicines10081985] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2022] [Revised: 08/11/2022] [Accepted: 08/12/2022] [Indexed: 12/21/2022] Open
Abstract
The transparency of the cornea along with its dense sensory innervation and resident leukocyte populations make it an ideal tissue to study interactions between the nervous and immune systems. The cornea is the most densely innervated tissue of the body and possesses both immune and vascular privilege, in part due to its unique repertoire of resident immune cells. Corneal nerves produce various neuropeptides that have a wide range of functions on immune cells. As research in this area expands, further insights are made into the role of neuropeptides and their immunomodulatory functions in the healthy and diseased cornea. Much remains to be known regarding the details of neuropeptide signaling and how it contributes to pathophysiology, which is likely due to complex interactions among neuropeptides, receptor isoform-specific signaling events, and the inflammatory microenvironment in disease. However, progress in this area has led to an increase in studies that have begun modulating neuropeptide activity for the treatment of corneal diseases with promising results, necessitating the need for a comprehensive review of the literature. This review focuses on the role of neuropeptides in maintaining the homeostasis of the ocular surface, alterations in disease settings, and the possible therapeutic potential of targeting these systems.
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Affiliation(s)
- Sudan Puri
- Center for Translational Ocular Immunology, Tufts Medical Center, Tufts University School of Medicine, Boston, MA 02111, USA
- Department of Ophthalmology, Tufts Medical Center, Tufts University School of Medicine, Boston, MA 02111, USA
| | - Brendan M. Kenyon
- Center for Translational Ocular Immunology, Tufts Medical Center, Tufts University School of Medicine, Boston, MA 02111, USA
- Program in Neuroscience, Graduate School of Biomedical Sciences, Tufts University, Boston, MA 02111, USA
| | - Pedram Hamrah
- Center for Translational Ocular Immunology, Tufts Medical Center, Tufts University School of Medicine, Boston, MA 02111, USA
- Department of Ophthalmology, Tufts Medical Center, Tufts University School of Medicine, Boston, MA 02111, USA
- Program in Neuroscience, Graduate School of Biomedical Sciences, Tufts University, Boston, MA 02111, USA
- Departments of Immunology and Neuroscience, Tufts University School of Medicine, Boston, MA 02111, USA
- Cornea Service, Tufts New England Eye Center, Boston, MA 02111, USA
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7
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Modulating the tachykinin: Role of substance P and neurokinin receptor expression in ocular surface disorders. Ocul Surf 2022; 25:142-153. [PMID: 35779793 DOI: 10.1016/j.jtos.2022.06.007] [Citation(s) in RCA: 23] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2022] [Revised: 06/21/2022] [Accepted: 06/24/2022] [Indexed: 01/19/2023]
Abstract
Substance P (SP) is a tachykinin expressed by various cells in the nervous and immune systems. SP is predominantly released by neurons and exerts its biological and immunological effects through the neurokinin receptors, primarily the neurokinin-1 receptor (NK1R). SP is essential for maintaining ocular surface homeostasis, and its reduced levels in disorders like diabetic neuropathy disrupt the corneal tissue. It also plays an essential role in promoting corneal wound healing by promoting the migration of keratocytes. In this review, we briefly discuss the structure, expression, and function of SP and its principal receptor NK1R. In addition, SP induces pro-inflammatory effects through autocrine or paracrine action on the immune cells in various ocular surface pathologies, including dry eye disease, herpes simplex virus keratitis, and Pseudomonas keratitis. We provide an in-depth review of the pathogenic role of SP in various ocular surface diseases and several new approaches developed to counter the immune-mediated effects of SP either through modulating its production or blocking its target receptor.
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8
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Ruiz-Viroga V, Urbanavicius J, Torterolo P, Lagos P. In vivo uptake of a fluorescent conjugate of melanin-concentrating hormone in the rat brain. J Chem Neuroanat 2021; 114:101959. [PMID: 33848617 DOI: 10.1016/j.jchemneu.2021.101959] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2020] [Revised: 04/03/2021] [Accepted: 04/08/2021] [Indexed: 11/16/2022]
Abstract
Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide synthesized by posterior hypothalamic and incerto-hypothalamic neurons that project throughout the central nervous system. The MCHergic system modulates several important functions such as feeding behavior, mood and sleep. MCH exerts its biological functions through interaction with the MCHR-1 receptor, the only functional receptor present in rodents. The internalization process of MCHR-1 triggered by MCH binding was described in vitro in non-neuronal heterologous systems with over-expression of MCHR-1. Reports of in vivo MCHR-1 internalization dynamics are scarce, however, this is an important process to explore based on the critical functions of the MCHergic system. We had previously determined that 60 min after intracerebroventricular (i.c.v.) microinjections of MCH conjugated with fluorophore rhodamine (R-MCH), the dorsal and median raphe nucleus presented R-MCH positive labeled neurons. In the present work, we further studied the in vivo uptake process focusing on the distribution and time-dependent pattern of R-MCH positive cells 10, 20 and 60 min (T10, T20 and T60, respectively) after i.c.v. microinjection of R-MCH. We also explored this uptake process to see whether it was receptor- and clathrin-dependent and examined the phenotype of R-MCH positive cells and their proximity to MCHergic fibers. We found a great number of R-MCH positive cells with high fluorescence intensity in the lateral septum, nucleus accumbens and hippocampus at T20 and T60 (but not at T10), while a lower number with low intensity was observed in the dorsal raphe nucleus. At T20, in rats pre-treated with a MCHR-1 antagonist (ATC-0175) or with phenylarsine oxide (PAO), a clathrin endocytosis inhibitor, a robust decrease (> 50 %) of R-MCH uptake occurred in these structures. The R-MCH positive cells were identified as neurons (NeuN positive, GFAP negative) and some MCHergic fibers run in the vicinities of them. We concluded that neurons localized at structures that were close to the ventricular surfaces could uptake R-MCH in vivo through a receptor-dependent and clathrin-mediated process. Our results support volume transmission of MCH through the cerebrospinal fluid to reach distant targets. Finally, we propose that R-MCH would be an effective tool to study MCH-uptake in vivo.
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Affiliation(s)
- Vicente Ruiz-Viroga
- Departamento de Fisiología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
| | - Jessika Urbanavicius
- Departamento de Neurofarmacología Experimental, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay
| | - Pablo Torterolo
- Departamento de Fisiología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
| | - Patricia Lagos
- Departamento de Fisiología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay.
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Ilie A, Boucher A, Park J, Berghuis AM, McKinney RA, Orlowski J. Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome. J Biol Chem 2020; 295:7075-7095. [PMID: 32277048 PMCID: PMC7242699 DOI: 10.1074/jbc.ra120.012614] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2020] [Revised: 04/07/2020] [Indexed: 12/15/2022] Open
Abstract
Genetic screening has identified numerous variants of the endosomal solute carrier family 9 member A6 (SLC9A6)/(Na+,K+)/H+ exchanger 6 (NHE6) gene that cause Christianson syndrome, a debilitating X-linked developmental disorder associated with a range of neurological, somatic, and behavioral symptoms. Many of these variants cause complete loss of NHE6 expression, but how subtler missense substitutions or nonsense mutations that partially truncate its C-terminal cytoplasmic regulatory domain impair NHE6 activity and endosomal function are poorly understood. Here, we describe the molecular and cellular consequences of six unique mutations located in the N-terminal cytoplasmic segment (A9S), the membrane ion translocation domain (L188P and G383D), and the C-terminal regulatory domain (E547*, R568Q, and W570*) of human NHE6 that purportedly cause disease. Using a heterologous NHE6-deficient cell expression system, we show that the biochemical, catalytic, and cellular properties of the A9S and R568Q variants were largely indistinguishable from those of the WT transporter, which obscured their disease significance. By contrast, the L188P, G383D, E547*, and W570* mutants exhibited variable deficiencies in biosynthetic post-translational maturation, membrane sorting, pH homeostasis in recycling endosomes, and cargo trafficking, and they also triggered apoptosis. These findings broaden our understanding of the molecular dysfunctions of distinct NHE6 variants associated with Christianson syndrome.
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Affiliation(s)
- Alina Ilie
- Department of Physiology, McGill University, Montreal, Quebec H3G 0B1, Canada
| | - Annie Boucher
- Department of Physiology, McGill University, Montreal, Quebec H3G 0B1, Canada
| | - Jaeok Park
- Department of Biochemistry, McGill University, Montreal, Quebec H3G 0B1, Canada
| | | | - R Anne McKinney
- Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec H3G 0B1, Canada
| | - John Orlowski
- Department of Physiology, McGill University, Montreal, Quebec H3G 0B1, Canada
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He X, Yan L, Wu Q, Zhang G, Zhou N. Ligand-dependent internalization of Bombyx mori tachykinin-related peptide receptor is regulated by PKC, GRK5 and β-arrestin2/BmKurtz. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2020; 1867:118690. [PMID: 32112783 DOI: 10.1016/j.bbamcr.2020.118690] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/11/2019] [Revised: 02/18/2020] [Accepted: 02/23/2020] [Indexed: 10/24/2022]
Abstract
Tachykinin signaling system is present in both vertebrates and invertebrates, and functions as neuromodulator responsible for the regulation of various physiological processes. In human, the internalization of G protein-coupled receptors has been extensively characterized; however, the insect GPCR internalization has been rarely investigated. Here, we constructed two expression vectors of Bombyx tachykinin-related peptide receptor (BmTKRPR) fused with Enhanced Green Fluorescent Protein (EGFP) at the C-terminal end for direct visualization of receptor expression, localization, and trafficking in cultured mammalian HEK293 and insect Sf21 cells. Our results demonstrated that agonist-activated BmTKRPR underwent rapid internalization in a dose-and time-dependent manner via a clathrin-dependent pathway in both HEK293 and Sf21 cells. Further investigation via RNAi or specific inhibitors, or co-immunoprecipitation demonstrated that agonist-induced BmTKRPR internalization was mediated by PKC, GRK5 and β-arrestin2/BmKurtz. In addition, we also observed that most of the internalized BmTKRP receptors were recycled to the cell surface via early endosomes upon peptide ligand removal. Our study provides the first in-depth information on mechanisms underlying insect TKRP receptor internalization and perhaps aids in the interpretation of the signaling in the regulation of physiological processes.
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Affiliation(s)
- Xiaobai He
- Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang 310058, China; College of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212018, China.
| | - Lili Yan
- Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang 310058, China
| | - Qi Wu
- College of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212018, China
| | - Guozheng Zhang
- Key Laboratory of Genetic Improvement of Sericulture, Ministry of Agriculture and Rural Affairs, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212018, China
| | - Naiming Zhou
- Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang 310058, China.
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Majkowska-Pilip A, Halik PK, Gniazdowska E. The Significance of NK1 Receptor Ligands and Their Application in Targeted Radionuclide Tumour Therapy. Pharmaceutics 2019; 11:E443. [PMID: 31480582 PMCID: PMC6781293 DOI: 10.3390/pharmaceutics11090443] [Citation(s) in RCA: 26] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2019] [Revised: 08/15/2019] [Accepted: 08/16/2019] [Indexed: 02/06/2023] Open
Abstract
To date, our understanding of the Substance P (SP) and neurokinin 1 receptor (NK1R) system shows intricate relations between human physiology and disease occurrence or progression. Within the oncological field, overexpression of NK1R and this SP/NK1R system have been implicated in cancer cell progression and poor overall prognosis. This review focuses on providing an update on the current state of knowledge around the wide spectrum of NK1R ligands and applications of radioligands as radiopharmaceuticals. In this review, data concerning both the chemical and biological aspects of peptide and nonpeptide ligands as agonists or antagonists in classical and nuclear medicine, are presented and discussed. However, the research presented here is primarily focused on NK1R nonpeptide antagonistic ligands and the potential application of SP/NK1R system in targeted radionuclide tumour therapy.
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Affiliation(s)
- Agnieszka Majkowska-Pilip
- Centre of Radiochemistry and Nuclear Chemistry, Institute of Nuclear Chemistry and Technology, Dorodna 16, 03-195 Warsaw, Poland.
| | - Paweł Krzysztof Halik
- Centre of Radiochemistry and Nuclear Chemistry, Institute of Nuclear Chemistry and Technology, Dorodna 16, 03-195 Warsaw, Poland
| | - Ewa Gniazdowska
- Centre of Radiochemistry and Nuclear Chemistry, Institute of Nuclear Chemistry and Technology, Dorodna 16, 03-195 Warsaw, Poland
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12
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Zhao P, Pattison LA, Jensen DD, Jimenez-Vargas NN, Latorre R, Lieu T, Jaramillo JO, Lopez-Lopez C, Poole DP, Vanner SJ, Schmidt BL, Bunnett NW. Protein kinase D and Gβγ mediate sustained nociceptive signaling by biased agonists of protease-activated receptor-2. J Biol Chem 2019; 294:10649-10662. [PMID: 31142616 DOI: 10.1074/jbc.ra118.006935] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2018] [Revised: 05/23/2019] [Indexed: 12/20/2022] Open
Abstract
Proteases sustain hyperexcitability and pain by cleaving protease-activated receptor-2 (PAR2) on nociceptors through distinct mechanisms. Whereas trypsin induces PAR2 coupling to Gαq, Gαs, and β-arrestins, cathepsin-S (CS) and neutrophil elastase (NE) cleave PAR2 at distinct sites and activate it by biased mechanisms that induce coupling to Gαs, but not to Gαq or β-arrestins. Because proteases activate PAR2 by irreversible cleavage, and activated PAR2 is degraded in lysosomes, sustained extracellular protease-mediated signaling requires mobilization of intact PAR2 from the Golgi apparatus or de novo synthesis of new receptors by incompletely understood mechanisms. We found here that trypsin, CS, and NE stimulate PAR2-dependent activation of protein kinase D (PKD) in the Golgi of HEK293 cells, in which PKD regulates protein trafficking. The proteases stimulated translocation of the PKD activator Gβγ to the Golgi, coinciding with PAR2 mobilization from the Golgi. Proteases also induced translocation of a photoconverted PAR2-Kaede fusion protein from the Golgi to the plasma membrane of KNRK cells. After incubation of HEK293 cells and dorsal root ganglia neurons with CS, NE, or trypsin, PAR2 responsiveness initially declined, consistent with PAR2 cleavage and desensitization, and then gradually recovered. Inhibitors of PKD, Gβγ, and protein translation inhibited recovery of PAR2 responsiveness. PKD and Gβγ inhibitors also attenuated protease-evoked mechanical allodynia in mice. We conclude that proteases that activate PAR2 by canonical and biased mechanisms stimulate PKD in the Golgi; PAR2 mobilization and de novo synthesis repopulate the cell surface with intact receptors and sustain nociceptive signaling by extracellular proteases.
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Affiliation(s)
- Peishen Zhao
- From the Monash Institute of Pharmaceutical Sciences and Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash University, Parkville, Victoria 3052, Australia
| | - Luke A Pattison
- From the Monash Institute of Pharmaceutical Sciences and Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash University, Parkville, Victoria 3052, Australia
| | - Dane D Jensen
- the Departments of Surgery and Pharmacology, Columbia University Vagelos College of Physicians and Surgeons, Columbia University, New York, New York 10032
| | - Nestor N Jimenez-Vargas
- the Gastrointestinal Diseases Research Unit, Division of Gastroenterology, Queen's University, Kingston, Ontario K7L 3N6, Canada, and
| | - Rocco Latorre
- the Departments of Surgery and Pharmacology, Columbia University Vagelos College of Physicians and Surgeons, Columbia University, New York, New York 10032
| | - TinaMarie Lieu
- From the Monash Institute of Pharmaceutical Sciences and Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash University, Parkville, Victoria 3052, Australia
| | - Josue O Jaramillo
- the Gastrointestinal Diseases Research Unit, Division of Gastroenterology, Queen's University, Kingston, Ontario K7L 3N6, Canada, and
| | - Cintya Lopez-Lopez
- the Gastrointestinal Diseases Research Unit, Division of Gastroenterology, Queen's University, Kingston, Ontario K7L 3N6, Canada, and
| | - Daniel P Poole
- From the Monash Institute of Pharmaceutical Sciences and Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash University, Parkville, Victoria 3052, Australia
| | - Stephen J Vanner
- the Gastrointestinal Diseases Research Unit, Division of Gastroenterology, Queen's University, Kingston, Ontario K7L 3N6, Canada, and
| | - Brian L Schmidt
- the Bluestone Center for Clinical Research, New York University College of Dentistry, New York, New York 10010
| | - Nigel W Bunnett
- the Departments of Surgery and Pharmacology, Columbia University Vagelos College of Physicians and Surgeons, Columbia University, New York, New York 10032,
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13
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Endothelin-converting enzyme-1 regulates glucagon-like peptide-1 receptor signalling and resensitisation. Biochem J 2019; 476:513-533. [PMID: 30626614 DOI: 10.1042/bcj20180853] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2018] [Revised: 01/07/2019] [Accepted: 01/08/2019] [Indexed: 02/08/2023]
Abstract
Following nutrient ingestion, glucagon-like peptide 1 (GLP-1) is secreted from intestinal L-cells and mediates anti-diabetic effects, most notably stimulating glucose-dependent insulin release from pancreatic β-cells but also inhibiting glucagon release, promoting satiety and weight reduction and potentially enhancing or preserving β-cell mass. These effects are mediated by the GLP-1 receptor (GLP-1R), which is a therapeutic target in type 2 diabetes. Although agonism at the GLP-1R has been well studied, desensitisation and resensitisation are perhaps less well explored. An understanding of these events is important, particularly in the design and use of novel receptor ligands. Here, using either HEK293 cells expressing the recombinant human GLP-1R or the pancreatic β-cell line, INS-1E with endogenous expressesion of the GLP-1R, we demonstrate GLP-1R desensitisation and subsequent resensitisation following removal of extracellular GLP-1 7-36 amide. Resensitisation is dependent on receptor internalisation, endosomal acidification and receptor recycling. Resensitisation is also regulated by endothelin-converting enzyme-1 (ECE-1) activity, most likely through proteolysis of GLP-1 in endosomes and the facilitation of GLP-1R dephosphorylation and recycling. Inhibition of ECE-1 activity also increases GLP-1-induced activation of extracellular signal-regulated kinase and generation of cAMP, suggesting processes dependent upon the lifetime of the internalised ligand-receptor complex.
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14
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Huang H, Zhang X, Fu X, Zhang X, Lang B, Xiang X, Hao W. Alcohol-induced conditioned place preference negatively correlates with anxiety-like behavior in adolescent mice: inhibition by a neurokinin-1 receptor antagonist. Psychopharmacology (Berl) 2018; 235:2847-2857. [PMID: 30054674 DOI: 10.1007/s00213-018-4976-7] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/31/2018] [Accepted: 07/17/2018] [Indexed: 01/23/2023]
Abstract
RATIONALE Although alcohol use disorder and anxiety disorders are highly comorbid in humans, controversy remains regarding whether anxiety predisposes individuals to alcohol reward, and the relationship with neurokinin-1 receptor (NK1R) is unclear. OBJECTIVES The objectives of the study are to investigate the association between anxiety-like behavior and alcohol-induced conditioned place preference (CPP) and to examine the effect of NK1R antagonist L-703,606 on this preference and levels of NK1R protein in different brain regions in adolescent mice. METHODS The anxiety-like behavior of adolescent male C57BL/6 mice was assessed using the elevated plus maze (EPM) test, and the animals were then allocated into high-anxiety mouse (HAM) and low-anxiety mouse (LAM) groups based on the percent of open arm time (OT%). After the reinforcement of ethanol was established by alcohol-induced CPP (2 g/kg), NK1R expression was quantified in the hippocampus, prefrontal cortex, and amygdala. Finally, the effect of L-703,606 (10 mg/kg) on the alcohol-induced CPP was examined. RESULTS LAM showed a greater ethanol preference (P = 0.004) and a higher level of NK1R protein in the hippocampus (P = 0.026) than HAM group. Interestingly, the CPP score positively correlated with OT% (r = 0.520, P = 0.016) and the level of NK1R protein (r = 0.476, P = 0.029) in the hippocampus. Moreover, L-703,606 attenuated alcohol-induced CPP (P < 0.001) in both groups. CONCLUSIONS The present results highlight the negative correlation between anxiety-like behavior and the propensity for alcohol and the critical role for NK1R in alcohol reward in adolescent mice. Importantly, the NK1R antagonist L-703,606 might be a promising therapeutic target for alcohol use disorder.
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Affiliation(s)
- Hui Huang
- Mental Health Institute of the Second Xiangya Hospital, The China National Clinical Research Center for Mental Health Disorders, National Technology Institute of Psychiatry, Key Laboratory of Psychiatry and Mental Health of Hunan Province, Central South University, Changsha, People's Republic of China.,Department of Psychiatry, The First Affiliated Hospital of Chongqing Medical University, Chongqing, People's Republic of China
| | - Xiaojie Zhang
- Mental Health Institute of the Second Xiangya Hospital, The China National Clinical Research Center for Mental Health Disorders, National Technology Institute of Psychiatry, Key Laboratory of Psychiatry and Mental Health of Hunan Province, Central South University, Changsha, People's Republic of China
| | - Xiaoya Fu
- Mental Health Institute of the Second Xiangya Hospital, The China National Clinical Research Center for Mental Health Disorders, National Technology Institute of Psychiatry, Key Laboratory of Psychiatry and Mental Health of Hunan Province, Central South University, Changsha, People's Republic of China
| | - Xiangyang Zhang
- Department of Psychiatry and Behavioral Sciences, The University of Texas Health Science Center at Houston, Houston, TX, USA
| | - Bing Lang
- Mental Health Institute of the Second Xiangya Hospital, The China National Clinical Research Center for Mental Health Disorders, National Technology Institute of Psychiatry, Key Laboratory of Psychiatry and Mental Health of Hunan Province, Central South University, Changsha, People's Republic of China
| | - Xiaojun Xiang
- Mental Health Institute of the Second Xiangya Hospital, The China National Clinical Research Center for Mental Health Disorders, National Technology Institute of Psychiatry, Key Laboratory of Psychiatry and Mental Health of Hunan Province, Central South University, Changsha, People's Republic of China.
| | - Wei Hao
- Mental Health Institute of the Second Xiangya Hospital, The China National Clinical Research Center for Mental Health Disorders, National Technology Institute of Psychiatry, Key Laboratory of Psychiatry and Mental Health of Hunan Province, Central South University, Changsha, People's Republic of China.
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15
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Lo CCW, Moosavi SM, Bubb KJ. The Regulation of Pulmonary Vascular Tone by Neuropeptides and the Implications for Pulmonary Hypertension. Front Physiol 2018; 9:1167. [PMID: 30190678 PMCID: PMC6116211 DOI: 10.3389/fphys.2018.01167] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2018] [Accepted: 08/03/2018] [Indexed: 12/20/2022] Open
Abstract
Pulmonary hypertension (PH) is an incurable, chronic disease of small pulmonary vessels. Progressive remodeling of the pulmonary vasculature results in increased pulmonary vascular resistance (PVR). This causes secondary right heart failure. PVR is tightly regulated by a range of pulmonary vasodilators and constrictors. Endothelium-derived substances form the basis of most current PH treatments. This is particularly the case for pulmonary arterial hypertension. The major limitation of current treatments is their inability to reverse morphological changes. Thus, there is an unmet need for novel therapies to reduce the morbidity and mortality in PH. Microvessels in the lungs are highly innervated by sensory C fibers. Substance P and calcitonin gene-related peptide (CGRP) are released from C-fiber nerve endings. These neuropeptides can directly regulate vascular tone. Substance P tends to act as a vasoconstrictor in the pulmonary circulation and it increases in the lungs during experimental PH. The receptor for substance P, neurokinin 1 (NK1R), mediates increased pulmonary pressure. Deactivation of NK1R with antagonists, or depletion of substance P prevents PH development. CGRP is a potent pulmonary vasodilator. CGRP receptor antagonists cause elevated pulmonary pressure. Thus, the balance of these peptides is crucial within the pulmonary circulation (Graphical Abstract). Limited progress has been made in understanding their impact on pulmonary pathophysiology. This is an intriguing area of investigation to pursue. It may lead to promising new candidate therapies to combat this fatal disease. This review provides a summary of the current knowledge in this area. It also explores possible future directions for neuropeptides in PH.
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Affiliation(s)
- Charmaine C. W. Lo
- Kolling Institute of Medical Research, University of Sydney, St Leonards, NSW, Australia
| | - Seyed M. Moosavi
- Kolling Institute of Medical Research, University of Sydney, St Leonards, NSW, Australia
- School of Life Sciences, University of Technology Sydney, Ultimo, NSW, Australia
| | - Kristen J. Bubb
- Kolling Institute of Medical Research, University of Sydney, St Leonards, NSW, Australia
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16
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Stoeber M, Jullié D, Lobingier BT, Laeremans T, Steyaert J, Schiller PW, Manglik A, von Zastrow M. A Genetically Encoded Biosensor Reveals Location Bias of Opioid Drug Action. Neuron 2018; 98:963-976.e5. [PMID: 29754753 DOI: 10.1016/j.neuron.2018.04.021] [Citation(s) in RCA: 215] [Impact Index Per Article: 30.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2017] [Revised: 03/26/2018] [Accepted: 04/17/2018] [Indexed: 11/16/2022]
Abstract
Opioid receptors (ORs) precisely modulate behavior when activated by native peptide ligands but distort behaviors to produce pathology when activated by non-peptide drugs. A fundamental question is how drugs differ from peptides in their actions on target neurons. Here, we show that drugs differ in the subcellular location at which they activate ORs. We develop a genetically encoded biosensor that directly detects ligand-induced activation of ORs and uncover a real-time map of the spatiotemporal organization of OR activation in living neurons. Peptide agonists produce a characteristic activation pattern initiated in the plasma membrane and propagating to endosomes after receptor internalization. Drugs produce a different activation pattern by additionally driving OR activation in the somatic Golgi apparatus and Golgi elements extending throughout the dendritic arbor. These results establish an approach to probe the cellular basis of neuromodulation and reveal that drugs distort the spatiotemporal landscape of neuronal OR activation.
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Affiliation(s)
- Miriam Stoeber
- Department of Psychiatry, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Damien Jullié
- Department of Psychiatry, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Braden T Lobingier
- Department of Psychiatry, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Toon Laeremans
- Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels, Belgium
| | - Jan Steyaert
- Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels, Belgium; VIB-VUB Center for Structural Biology, 1050 Brussels, Belgium
| | - Peter W Schiller
- Clinical Research Institute of Montreal, Montreal, QC H2W 1R7, Canada
| | - Aashish Manglik
- Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Anesthesia, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Mark von Zastrow
- Department of Psychiatry, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94143, USA.
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17
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Roelse M, Henquet MGL, Verhoeven HA, de Ruijter NCA, Wehrens R, van Lenthe MS, Witkamp RF, Hall RD, Jongsma MA. Calcium Imaging of GPCR Activation Using Arrays of Reverse Transfected HEK293 Cells in a Microfluidic System. SENSORS 2018; 18:s18020602. [PMID: 29462903 PMCID: PMC5855233 DOI: 10.3390/s18020602] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/06/2017] [Revised: 02/06/2018] [Accepted: 02/12/2018] [Indexed: 11/16/2022]
Abstract
Reverse-transfected cell arrays in microfluidic systems have great potential to perform large-scale parallel screening of G protein-coupled receptor (GPCR) activation. Here, we report the preparation of a novel platform using reverse transfection of HEK293 cells, imaging by stereo-fluorescence microscopy in a flowcell format, real-time monitoring of cytosolic calcium ion fluctuations using the fluorescent protein Cameleon and analysis of GPCR responses to sequential sample exposures. To determine the relationship between DNA concentration and gene expression, we analyzed cell arrays made with variable concentrations of plasmid DNA encoding fluorescent proteins and the Neurokinin 1 (NK1) receptor. We observed pronounced effects on gene expression of both the specific and total DNA concentration. Reverse transfected spots with NK1 plasmid DNA at 1% of total DNA still resulted in detectable NK1 activation when exposed to its ligand. By varying the GPCR DNA concentration in reverse transfection, the sensitivity and robustness of the receptor response for sequential sample exposures was optimized. An injection series is shown for an array containing the NK1 receptor, bitter receptor TAS2R8 and controls. Both receptors were exposed 14 times to alternating samples of two ligands. Specific responses remained reproducible. This platform introduces new opportunities for high throughput screening of GPCR libraries.
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Affiliation(s)
- Margriet Roelse
- BU Bioscience, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.
- Laboratory of Plant Physiology, Wageningen University and Research, 6708 PB Wageningen, The Netherlands.
| | - Maurice G L Henquet
- BU Bioscience, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.
| | - Harrie A Verhoeven
- BU Bioscience, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.
| | - Norbert C A de Ruijter
- Laboratory of Cell Biology, Wageningen University and Research, 6708 PB Wageningen, The Netherlands.
| | - Ron Wehrens
- BU Bioscience, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.
- BU Biometris, Wageningen University and Research, 6708 PB Wageningen, The Netherlands.
| | - Marco S van Lenthe
- BU Biometris, Wageningen University and Research, 6708 PB Wageningen, The Netherlands.
| | - Renger F Witkamp
- Human Nutrition and Health, Wageningen University and Research, Stippeneng 4, 6708 WE Wageningen, The Netherlands.
| | - Robert D Hall
- BU Bioscience, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.
- Laboratory of Plant Physiology, Wageningen University and Research, 6708 PB Wageningen, The Netherlands.
| | - Maarten A Jongsma
- BU Bioscience, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.
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18
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Roux BT, Bauer CC, McNeish AJ, Ward SG, Cottrell GS. The Role of Ubiquitination and Hepatocyte Growth Factor-Regulated Tyrosine Kinase Substrate in the Degradation of the Adrenomedullin Type I Receptor. Sci Rep 2017; 7:12389. [PMID: 28959041 PMCID: PMC5620052 DOI: 10.1038/s41598-017-12585-z] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2017] [Accepted: 09/06/2017] [Indexed: 01/27/2023] Open
Abstract
Calcitonin receptor-like receptor (CLR) and the receptor activity-modifying protein 2 (RAMP2) comprise a receptor for adrenomedullin (AM). Although it is known that AM induces internalization of CLR•RAMP2, little is known about the molecular mechanisms that regulate the trafficking of CLR•RAMP2. Using HEK and HMEC-1 cells, we observed that AM-induced activation of CLR•RAMP2 promoted ubiquitination of CLR. A mutant (CLRΔ9KR), lacking all intracellular lysine residues was functional and trafficked similar to the wild-type receptor, but was not ubiquitinated. Degradation of CLR•RAMP2 and CLRΔ9KR•RAMP2 was not dependent on the duration of AM stimulation or ubiquitination and occurred via a mechanism that was partially prevented by peptidase inhibitors. Degradation of CLR•RAMP2 was sensitive to overexpression of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), but not to HRS knockdown, whereas CLRΔ9KR•RAMP2 degradation was unaffected. Overexpression, but not knockdown of HRS, promoted hyperubiquitination of CLR under basal conditions. Thus, we propose a role for ubiquitin and HRS in the regulation of AM-induced degradation of CLR•RAMP2.
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Affiliation(s)
- Benoît T Roux
- Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath, BA2 7AY, UK
| | - Claudia C Bauer
- Cellular and Molecular Neuroscience, Reading School of Pharmacy, University of Reading, Reading, RG6 6UB, UK
| | - Alister J McNeish
- Cellular and Molecular Neuroscience, Reading School of Pharmacy, University of Reading, Reading, RG6 6UB, UK
| | - Stephen G Ward
- Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath, BA2 7AY, UK
| | - Graeme S Cottrell
- Cellular and Molecular Neuroscience, Reading School of Pharmacy, University of Reading, Reading, RG6 6UB, UK.
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19
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Fukui S, Ooyama N, Tamura J, Umar MA, Ishizuka T, Itami T, Miyoshi K, Sano T, Yamashita K. Interaction between maropitant and carprofen on sparing of the minimum alveolar concentration for blunting adrenergic response (MAC-BAR) of sevoflurane in dogs. J Vet Med Sci 2017; 79:502-508. [PMID: 28111373 PMCID: PMC5383168 DOI: 10.1292/jvms.15-0666] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Maropitant, a neurokinin-1 receptor antagonist, may provide analgesic effects by blocking pharmacological action of substance P. Carprofen is a non-steroidal
anti-inflammatory drug commonly used for pain control in dogs. The purpose of this study was to evaluate the effect of a combination of maropitant and carprofen
on the minimum alveolar concentration for blunting adrenergic response (MAC-BAR) of sevoflurane in dogs. Six healthy adult beagle dogs were anesthetized with
sevoflurane four times with a minimum of 7-day washout period. On each occasion, maropitant (1 mg/kg) alone, carprofen (4 mg/kg) alone, a combination of
maropitant (1 mg/kg) and carprofen (4 mg/kg), or saline (0.1 ml/kg) was subcutaneously administered at 1 hr prior to the first electrical
stimulation for the sevoflurane MAC-BAR determination. The sevoflurane MAC-BAR was significantly reduced by maropitant alone (2.88 ± 0.73%,
P=0.010), carprofen alone (2.96 ± 0.38%, P=0.016) and the combination (2.81 ± 0.51%, P=0.0003), compared with
saline (3.37 ± 0.56%). There was no significant difference in the percentage of MAC-BAR reductions between maropitant alone, carprofen alone and the
combination. The administration of maropitant alone and carprofen alone produced clinically significant sparing effects on the sevoflurane MAC-BAR in dogs.
However, the combination of maropitant and carprofen did not produce any additive effect on the sevoflurane MAC-BAR reduction. Anesthetic premedication with a
combination of maropitant and carprofen may not provide any further sparing effect on anesthetic requirement in dogs.
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Affiliation(s)
- Sho Fukui
- Department of Small Animal Clinical Sciences, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8591, Japan
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20
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Mashaghi A, Marmalidou A, Tehrani M, Grace PM, Pothoulakis C, Dana R. Neuropeptide substance P and the immune response. Cell Mol Life Sci 2016; 73:4249-4264. [PMID: 27314883 PMCID: PMC5056132 DOI: 10.1007/s00018-016-2293-z] [Citation(s) in RCA: 306] [Impact Index Per Article: 34.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2016] [Revised: 05/25/2016] [Accepted: 06/09/2016] [Indexed: 02/07/2023]
Abstract
Substance P is a peptide mainly secreted by neurons and is involved in many biological processes, including nociception and inflammation. Animal models have provided insights into the biology of this peptide and offered compelling evidence for the importance of substance P in cell-to-cell communication by either paracrine or endocrine signaling. Substance P mediates interactions between neurons and immune cells, with nerve-derived substance P modulating immune cell proliferation rates and cytokine production. Intriguingly, some immune cells have also been found to secrete substance P, which hints at an integral role of substance P in the immune response. These communications play important functional roles in immunity including mobilization, proliferation and modulation of the activity of immune cells. This review summarizes current knowledge of substance P and its receptors, as well as its physiological and pathological roles. We focus on recent developments in the immunobiology of substance P and discuss the clinical implications of its ability to modulate the immune response.
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Affiliation(s)
- Alireza Mashaghi
- Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA 02114 USA
| | - Anna Marmalidou
- Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA 02114 USA
| | - Mohsen Tehrani
- Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA 02114 USA
| | - Peter M. Grace
- Department of Psychology and Neuroscience, Center for Neuroscience, University of Colorado, Boulder, CO 80309 USA
| | - Charalabos Pothoulakis
- Division of Digestive Diseases, David Geffen School of Medicine, Inflammatory Bowel Disease Center, University of California, Los Angeles, Los Angeles, CA USA
| | - Reza Dana
- Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA 02114 USA
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21
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Liebick M, Schläger C, Oppermann M. Analysis of Chemokine Receptor Trafficking by Site-Specific Biotinylation. PLoS One 2016; 11:e0157502. [PMID: 27310579 PMCID: PMC4911081 DOI: 10.1371/journal.pone.0157502] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2016] [Accepted: 05/31/2016] [Indexed: 12/17/2022] Open
Abstract
Chemokine receptors undergo internalization and desensitization in response to ligand activation. Internalized receptors are either preferentially directed towards recycling pathways (e.g. CCR5) or sorted for proteasomal degradation (e.g. CXCR4). Here we describe a method for the analysis of receptor internalization and recycling based on specific Bir A-mediated biotinylation of an acceptor peptide coupled to the receptor, which allows a more detailed analysis of receptor trafficking compared to classical antibody-based detection methods. Studies on constitutive internalization of the chemokine receptors CXCR4 (12.1% ± 0.99% receptor internalization/h) and CCR5 (13.7% ± 0.68%/h) reveals modulation of these processes by inverse (TAK779; 10.9% ± 0.95%/h) or partial agonists (Met-CCL5; 15.6% ± 0.5%/h). These results suggest an actively driven internalization process. We also demonstrate the advantages of specific biotinylation compared to classical antibody detection during agonist-induced receptor internalization, which may be used for immunofluorescence analysis as well. Site-specific biotinylation may be applicable to studies on trafficking of transmembrane proteins, in general.
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MESH Headings
- Amides/pharmacology
- Animals
- Antibodies, Monoclonal/biosynthesis
- Antibodies, Monoclonal/chemistry
- Antibodies, Monoclonal/isolation & purification
- Basophils/cytology
- Basophils/drug effects
- Basophils/metabolism
- Biotin/chemistry
- Biotin/metabolism
- Biotinylation
- CCR5 Receptor Antagonists/pharmacology
- Carbon-Nitrogen Ligases/genetics
- Carbon-Nitrogen Ligases/metabolism
- Cell Line, Tumor
- Chemokine CCL5/pharmacology
- Escherichia coli Proteins/genetics
- Escherichia coli Proteins/metabolism
- Gene Expression
- Genetic Vectors/chemistry
- Genetic Vectors/metabolism
- Mice
- Protein Transport/drug effects
- Quaternary Ammonium Compounds/pharmacology
- Rats
- Receptors, CXCR4/antagonists & inhibitors
- Receptors, CXCR4/genetics
- Receptors, CXCR4/metabolism
- Receptors, CXCR5/antagonists & inhibitors
- Receptors, CXCR5/genetics
- Receptors, CXCR5/metabolism
- Recombinant Fusion Proteins/genetics
- Recombinant Fusion Proteins/metabolism
- Repressor Proteins/genetics
- Repressor Proteins/metabolism
- Transfection
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Affiliation(s)
- Marcel Liebick
- Department of Cellular and Molecular Immunology, University of Göttingen, Göttingen, Niedersachsen, Germany
| | - Christian Schläger
- Department of Cellular and Molecular Immunology, University of Göttingen, Göttingen, Niedersachsen, Germany
| | - Martin Oppermann
- Department of Cellular and Molecular Immunology, University of Göttingen, Göttingen, Niedersachsen, Germany
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Evidence of substance P autocrine circuitry that involves TNF-α, IL-6, and PGE2 in endogenous pyrogen-induced fever. J Neuroimmunol 2016; 293:1-7. [DOI: 10.1016/j.jneuroim.2016.01.016] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2015] [Revised: 01/11/2016] [Accepted: 01/25/2016] [Indexed: 11/23/2022]
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Wagner L, Wolf R, Zeitschel U, Rossner S, Petersén Å, Leavitt BR, Kästner F, Rothermundt M, Gärtner UT, Gündel D, Schlenzig D, Frerker N, Schade J, Manhart S, Rahfeld JU, Demuth HU, von Hörsten S. Proteolytic degradation of neuropeptide Y (NPY) from head to toe: Identification of novel NPY-cleaving peptidases and potential drug interactions in CNS and Periphery. J Neurochem 2015; 135:1019-37. [PMID: 26442809 DOI: 10.1111/jnc.13378] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2015] [Revised: 09/09/2015] [Accepted: 09/14/2015] [Indexed: 01/24/2023]
Abstract
The bioactivity of neuropeptide Y (NPY) is either N-terminally modulated with respect to receptor selectivity by dipeptidyl peptidase 4 (DP4)-like enzymes or proteolytic degraded by neprilysin or meprins, thereby abrogating signal transduction. However, neither the subcellular nor the compartmental differentiation of these regulatory mechanisms is fully understood. Using mass spectrometry, selective inhibitors and histochemistry, studies across various cell types, body fluids, and tissues revealed that most frequently DP4-like enzymes, aminopeptidases P, secreted meprin-A (Mep-A), and cathepsin D (CTSD) rapidly hydrolyze NPY, depending on the cell type and tissue under study. Novel degradation of NPY by cathepsins B, D, L, G, S, and tissue kallikrein could also be identified. The expression of DP4, CTSD, and Mep-A at the median eminence indicates that the bioactivity of NPY is regulated by peptidases at the interphase between the periphery and the CNS. Detailed ex vivo studies on human sera and CSF samples recognized CTSD as the major NPY-cleaving enzyme in the CSF, whereas an additional C-terminal truncation by angiotensin-converting enzyme could be detected in serum. The latter finding hints to potential drug interaction between antidiabetic DP4 inhibitors and anti-hypertensive angiotensin-converting enzyme inhibitors, while it ablates suspected hypertensive side effects of only antidiabetic DP4-inhibitors application. The bioactivity of neuropeptide Y (NPY) is either N-terminally modulated with respect to receptor selectivity by dipeptidyl peptidase 4 (DP4)-like enzymes or proteolytic degraded by neprilysin or meprins, thereby abrogating signal transduction. However, neither the subcellular nor the compartmental differentiation of these regulatory mechanisms is fully understood. Using mass spectrometry, selective inhibitors and histochemistry, studies across various cell types, body fluids, and tissues revealed that most frequently DP4-like enzymes, aminopeptidases P, secreted meprin-A (Mep-A), and cathepsin D (CTSD) rapidly hydrolyze NPY, depending on the cell type and tissue under study. Novel degradation of NPY by cathepsins B, D, L, G, S, and tissue kallikrein could also be identified. The expression of DP4, CTSD, and Mep-A at the median eminence indicates that the bioactivity of NPY is regulated by peptidases at the interphase between the periphery and the CNS. Detailed ex vivo studies on human sera and CSF samples recognized CTSD as the major NPY-cleaving enzyme in the CSF, whereas an additional C-terminal truncation by angiotensin-converting enzyme could be detected in serum. The latter finding hints to potential drug interaction between antidiabetic DP4 inhibitors and anti-hypertensive angiotensin-converting enzyme inhibitors, while it ablates suspected hypertensive side effects of only antidiabetic DP4-inhibitors application.
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Affiliation(s)
- Leona Wagner
- Deutschsprachige Selbsthilfegruppe für Alkaptonurie (DSAKU) e.V., Stuttgart, Germany.,Probiodrug AG, Halle, Germany.,Department of Experimental Therapy, Preclinical Experimental Center, Universitätsklinikum Erlangen, Erlangen, Germany
| | | | - Ulrike Zeitschel
- Paul-Flechsig-Institute for Brain Research, University of Leipzig, Leipzig, Germany
| | - Steffen Rossner
- Paul-Flechsig-Institute for Brain Research, University of Leipzig, Leipzig, Germany
| | - Åsa Petersén
- Translational Neuroendocrine Research Unit, Lund University, Lund, Sweden
| | - Blair R Leavitt
- Department of Medical Genetics, Centre for Molecular Medicine and Therapeutics, University of British Columbia and Children's and Women's Hospital, Vancouver, BC, Canada
| | - Florian Kästner
- Department of Psychiatry, University of Muenster, Muenster, Germany
| | - Matthias Rothermundt
- Department of Psychiatry, University of Muenster, Muenster, Germany.,St. Rochus-Hospital Telgte, Telgte, Germany
| | | | - Daniel Gündel
- Julius Bernstein Institute for Physiology, Martin Luther University of Halle-Wittenberg, Halle, Germany
| | - Dagmar Schlenzig
- Fraunhofer-Institute for Cell Therapy and Immunology, Department of Drug Design and Target Validation, Halle, Germany
| | - Nadine Frerker
- Department of Experimental Therapy, Preclinical Experimental Center, Universitätsklinikum Erlangen, Erlangen, Germany
| | - Jutta Schade
- Department of Experimental Therapy, Preclinical Experimental Center, Universitätsklinikum Erlangen, Erlangen, Germany
| | | | - Jens-Ulrich Rahfeld
- Fraunhofer-Institute for Cell Therapy and Immunology, Department of Drug Design and Target Validation, Halle, Germany
| | - Hans-Ulrich Demuth
- Fraunhofer-Institute for Cell Therapy and Immunology, Department of Drug Design and Target Validation, Halle, Germany
| | - Stephan von Hörsten
- Department of Experimental Therapy, Preclinical Experimental Center, Universitätsklinikum Erlangen, Erlangen, Germany
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Veya L, Piguet J, Vogel H. Single Molecule Imaging Deciphers the Relation between Mobility and Signaling of a Prototypical G Protein-coupled Receptor in Living Cells. J Biol Chem 2015; 290:27723-35. [PMID: 26363070 DOI: 10.1074/jbc.m115.666677] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2015] [Indexed: 01/10/2023] Open
Abstract
Lateral diffusion enables efficient interactions between membrane proteins, leading to signal transmission across the plasma membrane. An open question is how the spatiotemporal distribution of cell surface receptors influences the transmembrane signaling network. Here we addressed this issue by studying the mobility of a prototypical G protein-coupled receptor, the neurokinin-1 receptor, during its different phases of cellular signaling. Attaching a single quantum dot to individual neurokinin-1 receptors enabled us to follow with high spatial and temporal resolution over long time regimes the fate of individual receptors at the plasma membrane. Single receptor trajectories revealed a very heterogeneous mobility distribution pattern with diffusion constants ranging from 0.0005 to 0.1 μm(2)/s comprising receptors freely diffusing and others confined in 100-600-nm-sized membrane domains as well as immobile receptors. A two-dimensional representation of mobility and confinement resolved two major, broadly distributed receptor populations, one showing high mobility and low lateral restriction and the other showing low mobility and high restriction. We found that about 40% of the receptors in the basal state are already confined in membrane domains and are associated with clathrin. After stimulation with an agonist, an additional 30% of receptors became further confined. Using inhibitors of clathrin-mediated endocytosis, we found that the fraction of confined receptors at the basal state depends on the quantity of membrane-associated clathrin and is correlated to a significant decrease of the canonical pathway activity of the receptors. This shows that the high plasticity of receptor mobility is of central importance for receptor homeostasis and fine regulation of receptor activity.
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Affiliation(s)
- Luc Veya
- From the Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland
| | - Joachim Piguet
- From the Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland
| | - Horst Vogel
- From the Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland
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25
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Biological and Pharmacological Aspects of the NK1-Receptor. BIOMED RESEARCH INTERNATIONAL 2015; 2015:495704. [PMID: 26421291 PMCID: PMC4573218 DOI: 10.1155/2015/495704] [Citation(s) in RCA: 145] [Impact Index Per Article: 14.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/23/2014] [Revised: 04/19/2015] [Accepted: 04/25/2015] [Indexed: 12/11/2022]
Abstract
The neurokinin 1 receptor (NK-1R) is the main receptor for the tachykinin family of peptides. Substance P (SP) is the major mammalian ligand and the one with the highest affinity. SP is associated with multiple processes: hematopoiesis, wound healing, microvasculature permeability, neurogenic inflammation, leukocyte trafficking, and cell survival. It is also considered a mitogen, and it has been associated with tumorigenesis and metastasis. Tachykinins and their receptors are widely expressed in various human systems such as the nervous, cardiovascular, genitourinary, and immune system. Particularly, NK-1R is found in the nervous system and in peripheral tissues and are involved in cellular responses such as pain transmission, endocrine and paracrine secretion, vasodilation, and modulation of cell proliferation. It also acts as a neuromodulator contributing to brain homeostasis and to sensory neuronal transmission associated with depression, stress, anxiety, and emesis. NK-1R and SP are present in brain regions involved in the vomiting reflex (the nucleus tractus solitarius and the area postrema). This anatomical localization has led to the successful clinical development of antagonists against NK-1R in the treatment of chemotherapy-induced nausea and vomiting (CINV). The first of these antagonists, aprepitant (oral administration) and fosaprepitant (intravenous administration), are prescribed for high and moderate emesis.
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26
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Hothersall JD, Brown AJ, Dale I, Rawlins P. Can residence time offer a useful strategy to target agonist drugs for sustained GPCR responses? Drug Discov Today 2015; 21:90-96. [PMID: 26226643 DOI: 10.1016/j.drudis.2015.07.015] [Citation(s) in RCA: 55] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2015] [Revised: 06/24/2015] [Accepted: 07/21/2015] [Indexed: 01/28/2023]
Abstract
Residence time describes the how long a ligand is bound to its target, and is attracting interest in drug discovery as a potential means of improving clinical efficacy by increasing target coverage. This concept, as originally applied to antagonists, is more complicated for G-protein-coupled receptor (GPCR) agonists because of the transiency of receptor responses (via desensitization and internalization). However, in some cases sustained GPCR agonist responses have been observed, with evidence consistent with a role for slow binding kinetics. We propose a model to explain our understanding of how residence time and rebinding might influence sustained signaling by internalized receptors. We also highlight the anticipated benefit for drug discovery of fully understanding and exploiting these phenomena to target desirable receptor response profiles selectively.
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Affiliation(s)
| | | | - Ian Dale
- AstraZeneca, Discovery Sciences, Cambridge Science Park, Cambridge CB4 0WG, UK
| | - Philip Rawlins
- AstraZeneca, Discovery Sciences, Cambridge Science Park, Cambridge CB4 0WG, UK.
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27
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Muñoz M, Coveñas R, Esteban F, Redondo M. The substance P/NK-1 receptor system: NK-1 receptor antagonists as anti-cancer drugs. J Biosci 2015; 40:441-63. [DOI: 10.1007/s12038-015-9530-8] [Citation(s) in RCA: 68] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
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Mozaffari S, Erfani M, Beiki D, Johari Daha F, Kobarfard F, Balalaie S, Fallahi B. Synthesis and preliminary evaluation of a new (99m)tc labeled substance p analogue as a potential tumor imaging agent. IRANIAN JOURNAL OF PHARMACEUTICAL RESEARCH : IJPR 2015; 14:97-110. [PMID: 25561916 PMCID: PMC4277623] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Neurokinin 1 receptors (NK1R) are overexpressed on several types of important human cancer cells. Substance P (SP) is the most specific endogenous ligand known for NK1Rs. Accordingly,a new SP analogue was synthesized and evaluated for detection of NK1R positive tumors.[6-hydrazinopyridine-3-carboxylic acid (HYNIC)-Tyr(8)-Met(O)(11)-SP] was synthesized and radiolabeled with (99m)Tc using ethylenediamine-N,N'-diacetic acid (EDDA)and Tricine as coligands. Common physicochemical properties of radioconjugate were studied and in-vitro cell line biological tests were accomplished to determine the receptor mediated characteristics. In-vivo biodistribution in normal and tumor bearingnude mice was also assessed. The cold peptide was prepared in high purity (>99%) and radiolabeled with (99m)Tc at high specific activities (84-112GBq/µmol) with an acceptable labeling yield (>95%). The radioconjugate was stable in-vitro in the presence of human serum and showed 44% protein binding to human serumalbumin. In-vitro cell line studies on U373MG cells showed an acceptable uptake up to 4.91 ± 0.22% with the ratio of 60.21 ± 1.19% for its specific fraction and increasing specific internalization during 4 h. Receptor binding assays on U373MG cells indicated a mean Kd of 2.46 ± 0.43 nM and Bmax of 128925 ± 8145 sites/cell. In-vivo investigations determined the specific tumor uptake in 3.36 percent of injected dose per gram (%ID/g) for U373MG cells and noticeable accumulations of activity in the intestines and lung. Predominant renal excretion pathway was demonstrated. Therefore, this new radiolabeled peptide could be a promising radiotracer for detection of NK1R positive primary or secondary tumors.
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Affiliation(s)
- Saeed Mozaffari
- Department of Radiopharmacy, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.
| | - Mostafa Erfani
- Nuclear Science Research School, Nuclear Science and Technology Research Institute (NSTRI), Atomic Energy Organization of Iran (AEOI), Tehran, Iran.
| | - Davood Beiki
- Research Center for Nuclear Medicine, Tehran University of Medical Sciences, Tehran, Iran. ,E-mail:
| | - Fariba Johari Daha
- Nuclear Science Research School, Nuclear Science and Technology Research Institute (NSTRI), Atomic Energy Organization of Iran (AEOI), Tehran, Iran.
| | - Farzad Kobarfard
- Department of Medicinal Chemistry, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
| | - Saeed Balalaie
- Peptide Chemistry Research Center, K. N. Toosi University of Technology, Tehran, Iran.
| | - Babak Fallahi
- Research Center for Nuclear Medicine, Tehran University of Medical Sciences, Tehran, Iran.
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29
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Murphy SR, Oslund KL, Hyde DM, Miller LA, Van Winkle LS, Schelegle ES. Ozone-induced airway epithelial cell death, the neurokinin-1 receptor pathway, and the postnatal developing lung. Am J Physiol Lung Cell Mol Physiol 2014; 307:L471-81. [PMID: 25063800 PMCID: PMC4166783 DOI: 10.1152/ajplung.00324.2013] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2013] [Accepted: 07/21/2014] [Indexed: 11/22/2022] Open
Abstract
Children are uniquely susceptible to ozone because airway and lung growth continue for an extensive period after birth. Early-life exposure of the rhesus monkey to repeated ozone cycles results in region-specific disrupted airway/lung growth, but the mediators and mechanisms are poorly understood. Substance P (SP), neurokinin-1 receptor (NK-1R); and nuclear receptor Nur77 (NR4A1) are signaling pathway components involved in ozone-induced cell death. We hypothesize that acute ozone (AO) exposure during postnatal airway development disrupts SP/NK-1R/Nur77 pathway expression and that these changes correlate with increased ozone-induced cell death. Our objectives were to 1) spatially define the normal development of the SP/NK-1R/Nur77 pathway in conducting airways; 2) compare how postnatal age modulates responses to AO exposure; and 3) determine how concomitant, episodic ozone exposure modifies age-specific acute responses. Male infant rhesus monkeys were assigned at age 1 mo to two age groups, 2 or 6 mo, and then to one of three exposure subgroups: filtered air (FA), FA+AO (AO: 8 h/day × 2 days), or episodic biweekly ozone exposure cycles (EAO: 8 h/day × 5 days/14-day cycle+AO). O3 = 0.5 ppm. We found that 1) ozone increases SP/NK-1R/Nur77 pathway expression in conducting airways, 2) an ozone exposure cycle (5 days/cycle) delivered early at age 2 mo resulted in an airway that was hypersensitive to AO exposure at the end of 2 mo, and 3) continued episodic exposure (11 cycles) resulted in an airway that was hyposensitive to AO exposure at 6 mo. These observations collectively associate with greater overall inflammation and epithelial cell death, particularly in early postnatal (2 mo), distal airways.
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Affiliation(s)
- Shannon R Murphy
- Center for Health and the Environment, University of California-Davis, Davis, California
| | - Karen L Oslund
- California National Primate Research Center, University of California-Davis, Davis, California; and
| | - Dallas M Hyde
- California National Primate Research Center, University of California-Davis, Davis, California; and School of Veterinary Medicine, Department of Anatomy, Physiology and Cell Biology, University of California-Davis, Davis, California
| | - Lisa A Miller
- California National Primate Research Center, University of California-Davis, Davis, California; and School of Veterinary Medicine, Department of Anatomy, Physiology and Cell Biology, University of California-Davis, Davis, California
| | - Laura S Van Winkle
- Center for Health and the Environment, University of California-Davis, Davis, California; School of Veterinary Medicine, Department of Anatomy, Physiology and Cell Biology, University of California-Davis, Davis, California
| | - Edward S Schelegle
- California National Primate Research Center, University of California-Davis, Davis, California; and School of Veterinary Medicine, Department of Anatomy, Physiology and Cell Biology, University of California-Davis, Davis, California
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Muñoz M, Coveñas R. Involvement of substance P and the NK-1 receptor in pancreatic cancer. World J Gastroenterol 2014; 20:2321-2334. [PMID: 24605029 PMCID: PMC3942835 DOI: 10.3748/wjg.v20.i9.2321] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/29/2013] [Revised: 12/23/2013] [Accepted: 01/20/2014] [Indexed: 02/06/2023] Open
Abstract
Pancreatic cancer is the fourth leading cause of cancer related-death for both men and women and the 1- and 5-year relative survival rates are 25% and 6%, respectively. Thus, it is urgent to investigate new antitumor drugs to improve the survival of pancreatic cancer patients. The peptide substance P (SP) has a widespread distribution throughout the body. After binding to the neurokinin-1 (NK-1) receptor, SP regulates biological functions related to cancer, such as tumor cell proliferation, neoangiogenesis, the migration of tumor cells for invasion, infiltration and metastasis, and it exerts an antiapoptotic effects on tumor cells. It is known that the SP/NK-1 receptor system is involved in pancreatic cancer progression: (1) pancreatic cancer cells and samples express NK-1 receptors; (2) the NK-1 receptor is overexpressed in pancreatic cancer cells in comparison with non-tumor cells; (3) nanomolar concentrations of SP induce pancreatic cancer cell proliferation; (4) NK-1 receptor antagonists inhibit pancreatic cell proliferation in a concentration-dependent manner, at a certain concentration, these antagonists inhibit 100% of tumor cells; (5) this antitumor action is mediated through the NK-1 receptor, and tumor cells die by apoptosis; and (6) NK-1 receptor antagonists inhibit angiogenesis in pancreatic cancer xenografts. All these data suggest that the SP/NK-1 receptor system could play an important role in the development of pancreatic cancer; that the NK-1 receptor could be a new promising therapeutic target in pancreatic cancer, and that NK-1 receptor antagonists could improve the treatment of pancreatic cancer.
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31
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Steinhoff MS, von Mentzer B, Geppetti P, Pothoulakis C, Bunnett NW. Tachykinins and their receptors: contributions to physiological control and the mechanisms of disease. Physiol Rev 2014; 94:265-301. [PMID: 24382888 DOI: 10.1152/physrev.00031.2013] [Citation(s) in RCA: 457] [Impact Index Per Article: 41.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
The tachykinins, exemplified by substance P, are one of the most intensively studied neuropeptide families. They comprise a series of structurally related peptides that derive from alternate processing of three Tac genes and are expressed throughout the nervous and immune systems. Tachykinins interact with three neurokinin G protein-coupled receptors. The signaling, trafficking, and regulation of neurokinin receptors have also been topics of intense study. Tachykinins participate in important physiological processes in the nervous, immune, gastrointestinal, respiratory, urogenital, and dermal systems, including inflammation, nociception, smooth muscle contractility, epithelial secretion, and proliferation. They contribute to multiple diseases processes, including acute and chronic inflammation and pain, fibrosis, affective and addictive disorders, functional disorders of the intestine and urinary bladder, infection, and cancer. Neurokinin receptor antagonists are selective, potent, and show efficacy in models of disease. In clinical trials there is a singular success: neurokinin 1 receptor antagonists to treat nausea and vomiting. New information about the involvement of tachykinins in infection, fibrosis, and pruritus justifies further trials. A deeper understanding of disease mechanisms is required for the development of more predictive experimental models, and for the design and interpretation of clinical trials. Knowledge of neurokinin receptor structure, and the development of targeting strategies to disrupt disease-relevant subcellular signaling of neurokinin receptors, may refine the next generation of neurokinin receptor antagonists.
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Substance P differentially modulates firing rate of solitary complex (SC) neurons from control and chronic hypoxia-adapted adult rats. PLoS One 2014; 9:e88161. [PMID: 24516602 PMCID: PMC3917864 DOI: 10.1371/journal.pone.0088161] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2013] [Accepted: 01/03/2014] [Indexed: 11/19/2022] Open
Abstract
NK1 receptors, which bind substance P, are present in the majority of brainstem regions that contain CO2/H(+)-sensitive neurons that play a role in central chemosensitivity. However, the effect of substance P on the chemosensitive response of neurons from these regions has not been studied. Hypoxia increases substance P release from peripheral afferents that terminate in the caudal nucleus tractus solitarius (NTS). Here we studied the effect of substance P on the chemosensitive responses of solitary complex (SC: NTS and dorsal motor nucleus) neurons from control and chronic hypoxia-adapted (CHx) adult rats. We simultaneously measured intracellular pH and electrical responses to hypercapnic acidosis in SC neurons from control and CHx adult rats using the blind whole cell patch clamp technique and fluorescence imaging microscopy. Substance P significantly increased the basal firing rate in SC neurons from control and CHx rats, although the increase was smaller in CHx rats. However, substance P did not affect the chemosensitive response of SC neurons from either group of rats. In conclusion, we found that substance P plays a role in modulating the basal firing rate of SC neurons but the magnitude of the effect is smaller for SC neurons from CHx adult rats, implying that NK1 receptors may be down regulated in CHx adult rats. Substance P does not appear to play a role in modulating the firing rate response to hypercapnic acidosis of SC neurons from either control or CHx adult rats.
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Muñoz M, Coveñas R. Involvement of substance P and the NK-1 receptor in cancer progression. Peptides 2013; 48:1-9. [PMID: 23933301 DOI: 10.1016/j.peptides.2013.07.024] [Citation(s) in RCA: 107] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/03/2013] [Revised: 07/29/2013] [Accepted: 07/29/2013] [Indexed: 12/21/2022]
Abstract
Many data suggest the deep involvement of the substance P (SP)/neurokinin (NK)-1 receptor system in cancer: (1) Tumor cells express SP, NK-1 receptors and mRNA for the tachykinin NK-1 receptor; (2) Several isoforms of the NK-1 receptor are expressed in tumor cells; (3) the NK-1 receptor is involved in the viability of tumor cells; (4) NK-1 receptors are overexpressed in tumor cells in comparison with normal ones and malignant tissues express more NK-1 receptors than benign tissues; (5) Tumor cells expressing the most malignant phenotypes show an increased percentage of NK-1 receptor expression; (6) The expression of preprotachykinin A is increased in tumor cells in comparison with the levels found in normal cells; (7) SP induces the proliferation and migration of tumor cells and stimulates angiogenesis by increasing the proliferation of endothelial cells; (8) NK-1 receptor antagonists elicit the inhibition of tumor cell growth; (9) The specific antitumor action of NK-1 receptor antagonists on tumor cells occurs through the NK-1 receptor; (10) Tumor cell death is due to apoptosis; (11) NK-1 receptor antagonists inhibit the migration of tumor cells and neoangiogenesis. The NK-1 receptor is a therapeutic target in cancer and NK-1 receptor antagonists could be considered as broad-spectrum antitumor drugs for the treatment of cancer. It seems that a common mechanism for cancer cell proliferation mediated by SP and the NK-1 receptor is triggered, as well as a common mechanism exerted by NK-1 receptor antagonists on tumor cells, i.e. apoptosis.
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Affiliation(s)
- Miguel Muñoz
- Virgen del Rocío University Hospital, Research Laboratory on Neuropeptides (IBIS), Sevilla, Spain.
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Roelse M, de Ruijter NC, Vrouwe EX, Jongsma MA. A generic microfluidic biosensor of G protein-coupled receptor activation—monitoring cytoplasmic [Ca2+] changes in human HEK293 cells. Biosens Bioelectron 2013; 47:436-44. [DOI: 10.1016/j.bios.2013.03.065] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2013] [Revised: 03/22/2013] [Accepted: 03/26/2013] [Indexed: 01/08/2023]
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Marked Effects of Tachykinin in Myositis Both in the Experimental Side and Contralaterally: Studies on NK-1 Receptor Expressions in an Animal Model. ISRN INFLAMMATION 2013; 2013:907821. [PMID: 24049666 PMCID: PMC3765760 DOI: 10.1155/2013/907821] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/28/2012] [Accepted: 12/18/2012] [Indexed: 01/16/2023]
Abstract
Muscle injury and inflammation (myositis) in a rabbit model of an unilateral muscle overuse were examined. It is unknown if the tachykinin system has a functional role in this situation. In this study, therefore, the neurokinin-1 receptor (NK-1R) expression patterns were evaluated. White blood cells, nerve fascicles, fine nerve fibers, and blood vessel walls in myositis areas showed NK-1R immunoreaction. NK-1R mRNA reactions were observable for white blood cells and blood vessel walls of these areas. NK-1R immunoreaction and NK-1R mRNA reactions were also seen for muscle fibers showing degenerative and regenerative features. There were almost no NK-1R immunoreactions in normal muscle tissue. Interestingly, marked NK-1R expressions were seen for myositis areas of both the experimental side and the contralateral nonexperimental side. EIA analyses showed that the concentration of substance P in the muscle tissue was clearly increased bilaterally at the experimental end stage, as compared to the situation for normal muscle tissue. These observations show that the tachykinin system is very much involved in the processes that occur in muscle injury/myositis. The effects can be related to proinflammatory effects and/or tissue repair. The fact that there are also marked NK-1R expressions contralaterally indicate that the tachykinin system has crossover effects.
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36
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Intranasally administered neuropeptide S (NPS) exerts anxiolytic effects following internalization into NPS receptor-expressing neurons. Neuropsychopharmacology 2012; 37:1323-37. [PMID: 22278093 PMCID: PMC3327839 DOI: 10.1038/npp.2011.317] [Citation(s) in RCA: 67] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Experiments in rodents revealed neuropeptide S (NPS) to constitute a potential novel treatment option for anxiety diseases such as panic and post-traumatic stress disorder. However, both its cerebral target sites and the molecular underpinnings of NPS-mediated effects still remain elusive. By administration of fluorophore-conjugated NPS, we pinpointed NPS target neurons in distinct regions throughout the entire brain. We demonstrated their functional relevance in the hippocampus. In the CA1 region, NPS modulates synaptic transmission and plasticity. NPS is taken up into NPS receptor-expressing neurons by internalization of the receptor-ligand complex as we confirmed by subsequent cell culture studies. Furthermore, we tracked internalization of intranasally applied NPS at the single-neuron level and additionally demonstrate that it is delivered into the mouse brain without losing its anxiolytic properties. Finally, we show that NPS differentially modulates the expression of proteins of the glutamatergic system involved inter alia in synaptic plasticity. These results not only enlighten the path of NPS in the brain, but also establish a non-invasive method for NPS administration in mice, thus strongly encouraging translation into a novel therapeutic approach for pathological anxiety in humans.
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Rosso M, Muñoz M, Berger M. The role of neurokinin-1 receptor in the microenvironment of inflammation and cancer. ScientificWorldJournal 2012; 2012:381434. [PMID: 22545017 PMCID: PMC3322385 DOI: 10.1100/2012/381434] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2011] [Accepted: 11/20/2011] [Indexed: 12/11/2022] Open
Abstract
The recent years have witnessed an exponential increase in cancer research, leading to a considerable investment in the field. However, with few exceptions, this effort has not yet translated into a better overall prognosis for patients with cancer, and the search for new drug targets continues. After binding to the specific neurokinin-1 (NK-1) receptor, the peptide substance P (SP), which is widely distributed in both the central and peripheral nervous systems, triggers a wide variety of functions. Antagonists against the NK-1 receptor are safe clinical drugs that are known to have anti-inflammatory, analgesic, anxiolytic, antidepressant, and antiemetic effects. Recently, it has become apparent that SP can induce tumor cell proliferation, angiogenesis, and migration via the NK-1 receptor, and that the SP/NK-1 receptor complex is an integral part of the microenvironment of inflammation and cancer. Therefore, the use of NK-1 receptor antagonists as a novel and promising approach for treating patients with cancer is currently under intense investigation. In this paper, we evaluate the recent scientific developments regarding this receptor system, its role in the microenvironment of inflammation and cancer, and its potentials and pitfalls for the usage as part of modern anticancer strategies.
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Affiliation(s)
- Marisa Rosso
- Research Laboratory on Neuropeptides, Hospital Infantil Universitario Virgen del Rocío, Avenida Manuel Siurot s/n, 41013 Seville, Spain.
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Pelayo JC, Poole DP, Steinhoff M, Cottrell GS, Bunnett NW. Endothelin-converting enzyme-1 regulates trafficking and signalling of the neurokinin 1 receptor in endosomes of myenteric neurones. J Physiol 2011; 589:5213-30. [PMID: 21878523 DOI: 10.1113/jphysiol.2011.214452] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022] Open
Abstract
Neuropeptide signalling at the plasma membrane is terminated by neuropeptide degradation by cell-surface peptidases, and by β-arrestin-dependent receptor desensitization and endocytosis. However, receptors continue to signal from endosomes by β-arrestin-dependent processes, and endosomal sorting mediates recycling and resensitization of plasma membrane signalling. The mechanisms that control signalling and trafficking of receptors in endosomes are poorly defined. We report a major role for endothelin-converting enzyme-1 (ECE-1) in controlling substance P (SP) and the neurokinin 1 receptor (NK(1)R) in endosomes of myenteric neurones. ECE-1 mRNA and protein were expressed by myenteric neurones of rat and mouse intestine. SP (10 nM, 10 min) induced interaction of NK(1)R and β-arrestin at the plasma membrane, and the SP-NK(1)R-β-arrestin signalosome complex trafficked by a dynamin-mediated mechanism to ECE-1-containing early endosomes, where ECE-1 can degrade SP. After 120 min, NK(1)R recycled from endosomes to the plasma membrane. ECE-1 inhibitors (SM-19712, PD-069185) and the vacuolar H(+)ATPase inhibitor bafilomycin A(1), which prevent endosomal SP degradation, suppressed NK(1)R recycling by >50%. Preincubation of neurones with SP (10 nM, 5 min) desensitized Ca(2+) transients to a second SP challenge after 10 min, and SP signals resensitized after 60 min. SM-19712 inhibited NK(1)R resensitization by >90%. ECE-1 inhibitors also caused sustained SP-induced activation of extracellular signal-regulated kinases, consistent with stabilization of the SP-NK(1)R-β-arrestin signalosome. By degrading SP and destabilizing endosomal signalosomes, ECE-1 has a dual role in controlling endocytic signalling and trafficking of the NK(1)R: promoting resensitization of G protein-mediated plasma membrane signalling, and terminating β-arrestin-mediated endosomal signalling.
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Affiliation(s)
- Juan-Carlos Pelayo
- Department of Surgery, University of California, San Francisco, San Francisco, CA 94143-0660, USA
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Murphy JE, Roosterman D, Cottrell GS, Padilla BE, Feld M, Brand E, Cedron WJ, Bunnett NW, Steinhoff M. Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor. Am J Physiol Cell Physiol 2011; 301:C780-91. [PMID: 21795521 DOI: 10.1152/ajpcell.00096.2011] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
Activated G protein-coupled receptors (GPCRs) are phosphorylated and interact with β-arrestins, which mediate desensitization and endocytosis. Endothelin-converting enzyme-1 (ECE-1) degrades neuropeptides in endosomes and can promote recycling. Although endocytosis, dephosphorylation, and recycling are accepted mechanisms of receptor resensitization, a large proportion of desensitized receptors can remain at the cell surface. We investigated whether reactivation of noninternalized, desensitized (phosphorylated) receptors mediates resensitization of the substance P (SP) neurokinin 1 receptor (NK(1)R). Herein, we report a novel mechanism of resensitization by which protein phosphatase 2A (PP2A) is recruited to dephosphorylate noninternalized NK(1)R. A desensitizing concentration of SP reduced cell-surface SP binding sites by only 25%, and SP-induced Ca(2+) signals were fully resensitized before cell-surface binding sites started to recover, suggesting resensitization of cell-surface-retained NK(1)R. SP induced association of β-arrestin1 and PP2A with noninternalized NK(1)R. β-Arrestin1 small interfering RNA knockdown prevented SP-induced association of cell-surface NK(1)R with PP2A, indicating that β-arrestin1 mediates this interaction. ECE-1 inhibition, by trapping β-arrestin1 in endosomes, also impeded SP-induced association of cell-surface NK(1)R with PP2A. Resensitization of NK(1)R signaling required both PP2A and ECE-1 activity. Thus, after stimulation with SP, PP2A interacts with noninternalized NK(1)R and mediates resensitization. PP2A interaction with NK(1)R requires β-arrestin1. ECE-1 promotes this process by releasing β-arrestin1 from NK(1)R in endosomes. These findings represent a novel mechanism of PP2A- and ECE-1-dependent resensitization of GPCRs.
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Affiliation(s)
- Jane E Murphy
- Department of Surgery, University of California, San Francisco, 94143-0660, USA
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40
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Malherbe P, Knoflach F, Hernandez MC, Hoffmann T, Schnider P, Porter RH, Wettstein JG, Ballard TM, Spooren W, Steward L. Characterization of RO4583298 as a novel potent, dual antagonist with in vivo activity at tachykinin NK₁ and NK₃ receptors. Br J Pharmacol 2011; 162:929-46. [PMID: 21039418 DOI: 10.1111/j.1476-5381.2010.01096.x] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
BACKGROUND AND PURPOSE Clinical results of osanetant and talnetant (selective-NK₃ antagonists) indicate that blocking the NK₃ receptor could be beneficial for the treatment of schizophrenia. The objective of this study was to characterize the in vitro and in vivo properties of a novel dual NK₁/NK₃ antagonist, RO4583298 (2-phenyl-N-(pyridin-3-yl)-N-methylisobutyramide derivative). EXPERIMENTAL APPROACH RO4583298 in vitro pharmacology was investigated using radioligand binding ([³H]-SP, [³H]-osanetant, [³H]-senktide), [³H]-inositol-phosphate accumulation Schild analysis (SP- or [MePhe⁷]-NKB-induced) and electrophysiological studies in guinea-pig substantia nigra pars compacta (SNpc). The in vivo activity of RO4583298 was assessed using reversal of GR73632-induced foot tapping in gerbils (GFT; NK₁) and senktide-induced tail whips in mice (MTW; NK₃). KEY RESULTS RO4583298 has a high-affinity for NK₁ (human and gerbil) and NK₃ (human, cynomolgus monkey, gerbil and guinea-pig) receptors and behaves as a pseudo-irreversible antagonist. Unusually it binds with high-affinity to mouse and rat NK₃, yet with a partial non-competitive mode of antagonism. In guinea-pig SNpc, RO4583298 inhibited the senktide-induced potentiation of spontaneous activity of dopaminergic neurones with an apparent non-competitive mechanism of action. RO4583298 (p.o.) robustly blocked the GFT response, and inhibited the MTW. CONCLUSIONS AND IMPLICATIONS RO4583298 is a high-affinity, non-competitive, long-acting in vivo NK₁/NK₃ antagonist; hence providing a useful in vitro and in vivo pharmacological tool to investigate the roles of NK₁ and NK₃ receptors in psychiatric disorders.
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Affiliation(s)
- P Malherbe
- Discovery Research CNS, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
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Lessard A, Coleman CG, Pickel VM. Chronic intermittent hypoxia reduces neurokinin-1 (NK(1)) receptor density in small dendrites of non-catecholaminergic neurons in mouse nucleus tractus solitarius. Exp Neurol 2010; 223:634-44. [PMID: 20206166 PMCID: PMC2864350 DOI: 10.1016/j.expneurol.2010.02.013] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2009] [Revised: 02/18/2010] [Accepted: 02/21/2010] [Indexed: 12/16/2022]
Abstract
Chronic intermittent hypoxia (CIH) is a frequent concomitant of sleep apnea, which can increase sympathetic nerve activity through mechanisms involving chemoreceptor inputs to the commissural nucleus of the solitary tract (cNTS). These chemosensory inputs co-store glutamate and substance P (SP), an endogenous ligand for neurokinin-1 (NK(1)) receptors. Acute hypoxia results in internalization of NK(1) receptors, suggesting that CIH also may affect the subcellular distribution of NK(1) receptors in subpopulations of cNTS neurons, some of which may express tyrosine hydroxylase, the rate-limiting enzyme for catecholamine synthesis (TH). To test this hypothesis, we examined dual immunolabeling for the NK(1) receptor and TH in the cNTS of male mice subjected to 10days or 35days of CIH or intermittent air. Electron microscopy revealed that NK(1) receptors and TH were almost exclusively localized within separate somatodendritic profiles in cNTS of control mice. In dendrites, immunogold particles identifying NK(1) receptors were prevalent in the cytoplasm and on the plasmalemmal surface. Compared with controls, CIH produced a significant region-specific decrease in the cytoplasmic (10 and 35days, P<0.05, unpaired Student t-test) and extrasynaptic plasmalemmal (35days, P<0.01, unpaired Student t-test) density of NK(1) immunogold particles exclusively in small (<0.1microm) dendrites without TH immunoreactivity. These results suggest that CIH produces a duration-dependent reduction in the availability of NK(1) receptors preferentially in small dendrites of non-catecholaminergic neurons in the cNTS. The implications of our findings are discussed with respect to their potential involvement in the slowly developing hypertension seen in sleep apnea patients.
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Affiliation(s)
- Andrée Lessard
- Division of Neurobiology, Department of Neurology and Neuroscience, Weill-Cornell Medical College of Cornell University, New York, NY 10021, USA
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Li G, Shi Y, Huang H, Zhang Y, Wu K, Luo J, Sun Y, Lu J, Benovic JL, Zhou N. Internalization of the human nicotinic acid receptor GPR109A is regulated by G(i), GRK2, and arrestin3. J Biol Chem 2010; 285:22605-18. [PMID: 20460384 DOI: 10.1074/jbc.m109.087213] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Nicotinic acid (niacin) has been widely used as a favorable lipid-lowering drug for several decades, and the orphan G protein-coupled receptor GPR109A has been identified to be a receptor for niacin. Mechanistic investigations have shown that as a G(i)-coupled receptor, GPR109A inhibits adenylate cyclase activity upon niacin activation, thereby inhibiting free fatty acid liberation. However, the underlying molecular mechanisms that regulate signaling and internalization of GPR109A remain largely unknown. To further characterize GPR109A internalization, we made a construct to express GPR109A fused with enhanced green fluorescent protein (EGFP) at its carboxyl-terminal end. In stable GPR109A-EGFP-expressing HEK-293 cells, GPR109A-EGFP was mainly localized at the plasma membrane and was rapidly internalized in a dose- and time-dependent manner upon agonist stimulation. GPR109A internalization was completely blocked by hypertonic sucrose, indicating that GPR109A internalizes via the clathrin-coated pit pathway. Further investigation demonstrated that internalized GPR109A was recycled to the cell surface after the removal of agonist, and recycling of the internalized receptors was not blocked by treatment with acidotropic agents, NH(4)Cl and monensin. Pertussis toxin pretreatment not only inhibited forskolin-induced cAMP accumulation and intracellular Ca(2+) mobilization; it also significantly attenuated agonist-promoted GPR109A internalization. Moreover, RNA interference experiments showed that knockdown of GRK2 (G protein-coupled receptor kinase 2) and arrestin3 expression significantly impaired receptor internalization. Taken together, these results indicate that the agonist-induced internalization of GPR109A receptors is regulated by GRK2 and arrestin3 in a pertussis toxin-sensitive manner and that internalized receptor recycling is independent of endosomal acidification.
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Affiliation(s)
- Guo Li
- Institute of Biochemistry, College of Life Science, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang 310058, China
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Palmquist AM, Pissiota A, Frans O, Ahs F, Långström B, Bergström M, Fredrikson M. Comment on "Decreased Neurokinin-1 (Substance P) Receptor Binding in Patients with Panic Disorder: Positron Emission Tomographic Study With [(18)F]SPA-RQ". Biol Psychiatry 2010; 67:e25-6. [PMID: 19854434 DOI: 10.1016/j.biopsych.2009.08.037] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/26/2009] [Accepted: 08/26/2009] [Indexed: 10/20/2022]
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Linnman C, Appel L, Furmark T, Söderlund A, Gordh T, Långström B, Fredrikson M. Ventromedial prefrontal neurokinin 1 receptor availability is reduced in chronic pain. Pain 2010; 149:64-70. [PMID: 20137858 DOI: 10.1016/j.pain.2010.01.008] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2009] [Revised: 12/30/2009] [Accepted: 01/12/2010] [Indexed: 11/19/2022]
Abstract
Neurokinin 1 (NK1) receptors are involved in pain and anxiety behaviors in animals, but little is known about central alterations in this receptor system in human pain. With positron emission tomography, using a [11]-Carbon labeled NK1 receptor antagonist, we demonstrate attenuated NK1 receptor availability in frontal, insular and cingulate cortex, as well as the hippocampus, amygdala and the periaqueductal gray area in patients with chronic pain. The reduced availability was most pronounced in the ventromedial prefrontal cortex (vmPFC), where attenuations correlated to measures of fear and avoidance of movement. Further, vmPFC NK1 levels also displayed opposing influences in patients as compared to controls on regional cerebral blood flow in the anterior cingulate. We conclude that the central NK1 receptor system is altered in human chronic pain. The results suggest that NK1 receptors in the vmPFC modulate motor inhibition, and contribute to fear and avoidance of movement.
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Affiliation(s)
- Clas Linnman
- Department of Psychology, Uppsala University, Uppsala, Sweden Uppsala Imanet AB, GE Healthcare, Uppsala, Sweden Department of Physiotherapy, School of Health, Care and Social Welfare, Mälardalen University, Västerås, Sweden Section of Physiotherapy, Department of Neuroscience, Uppsala University, Sweden Laboratory of Pain Research, Department of Surgical Sciences, Division of Anaesthesiology and Intensive Care Medicine, Uppsala University Hospital, Sweden Department of Biochemistry and Organic Chemistry, Uppsala University, Sweden Neuropsychopharmacology Section, Faculty of Medicine, Imperial College, London, UK
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Sio SWS, Moochhala S, Lu J, Bhatia M. Early protection from burn-induced acute lung injury by deletion of preprotachykinin-A gene. Am J Respir Crit Care Med 2010; 181:36-46. [PMID: 19797759 DOI: 10.1164/rccm.200907-1073oc] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
RATIONALE Burn-induced acute lung injury (ALI) is a common clinical disorder associated with high mortality even in the absence of inhalational injury. Identification of endogenous triggers that mediate the early onset of remote ALI after burn represents an important goal but remains poorly defined. OBJECTIVES We investigated the role of proinflammatory neuropeptide, substance P (SP), in instigating remote ALI and its effects on respiratory function early after severe local burn injury. METHODS A 30% total body surface area full-thickness burn was induced in wild-type (WT) mice, preprotachykinin-A (PPT-A) gene deficient mice, which encodes for SP, and PPT-A(-/-) mice challenged with exogenous SP, followed by ALI and lung function analysis. MEASUREMENTS AND MAIN RESULTS Endogenous SP production was heightened in burn-injured WT mice, which induced significant elevation of proinflammatory cytokines, chemokines, and endothelial adhesion molecules concurrent with disruption of pulmonary permeability barrier, excessive neutrophil infiltration, and severe ALI. Additionally, decreased neutral endopeptidase and elevated matrix metalloproteinase-9 were evident. Notably, disruption of respiratory function demonstrates a critical role of SP in lungs after burn. These effects were significantly attenuated in PPT-A(-/-) mice, whereas the exogenous administration of SP to PPT-A(-/-) mice restored the inflammatory response and ALI. Furthermore, analysis of neurokinin-1-receptor (NK1R), to which SP binds preferentially, revealed that SP in conjunction with burn injury regulates NK1R expression. CONCLUSIONS We show that the absence of a single endogenous factor, SP, significantly provides early protection against burn-induced ALI in mice with marked improvement in respiratory function. Thereby, the blockade of SP may be beneficial in preventing early inflammation and ALI in patients with critical burn injuries.
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Affiliation(s)
- Selena W S Sio
- Department of Pharmacology, National University of Singapore, Singapore
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Yu YJ, Arttamangkul S, Evans CJ, Williams JT, von Zastrow M. Neurokinin 1 receptors regulate morphine-induced endocytosis and desensitization of mu-opioid receptors in CNS neurons. J Neurosci 2009; 29:222-33. [PMID: 19129399 PMCID: PMC2775560 DOI: 10.1523/jneurosci.4315-08.2009] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2008] [Revised: 10/29/2008] [Accepted: 11/28/2008] [Indexed: 01/01/2023] Open
Abstract
mu-Opioid receptors (MORs) are G-protein-coupled receptors (GPCRs) that mediate the physiological effects of endogenous opioid neuropeptides and opiate drugs such as morphine. MORs are coexpressed with neurokinin 1 receptors (NK1Rs) in several regions of the CNS that control opioid dependence and reward. NK1R activation affects opioid reward specifically, however, and the cellular basis for this specificity is unknown. We found that ligand-induced activation of NK1Rs produces a cell-autonomous and nonreciprocal inhibition of MOR endocytosis induced by diverse opioids. Studies using epitope-tagged receptors expressed in cultured striatal neurons and a neuroblastoma cell model indicated that this heterologous regulation is mediated by NK1R-dependent sequestration of arrestins on endosome membranes. First, endocytic inhibition mediated by wild-type NK1Rs was overcome in cells overexpressing beta-arrestin2, a major arrestin isoform expressed in striatum. Second, NK1R activation promoted sequestration of beta-arrestin2 on endosomes, whereas MOR activation did not. Third, heterologous inhibition of MOR endocytosis was prevented by mutational disruption of beta-arrestin2 sequestration by NK1Rs. NK1R-mediated regulation of MOR trafficking was associated with reduced opioid-induced desensitization of adenylyl cyclase signaling in striatal neurons. Furthermore, heterologous regulation of MOR trafficking was observed in both amygdala and locus ceruleus neurons that naturally coexpress these receptors. These results identify a cell-autonomous mechanism that may underlie the highly specific effects of NK1R on opioid signaling and suggest, more generally, that receptor-specific trafficking of arrestins may represent a fundamental mechanism for coordinating distinct GPCR-mediated signals at the level of individual CNS neurons.
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MESH Headings
- Analgesics, Opioid/pharmacology
- Animals
- Biotinylation/methods
- Cell Line, Tumor
- Corpus Striatum/cytology
- Cyclic AMP/metabolism
- Dose-Response Relationship, Drug
- Embryo, Mammalian
- Endocytosis/drug effects
- Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacology
- Enkephalin, Methionine/pharmacology
- Female
- Locus Coeruleus/cytology
- Mice
- Morphine/pharmacology
- Mutation/genetics
- Neuroblastoma
- Neurons/drug effects
- Pregnancy
- Rats
- Rats, Sprague-Dawley
- Receptors, Neurokinin-1/physiology
- Receptors, Opioid, mu/genetics
- Receptors, Opioid, mu/metabolism
- Substance P/pharmacology
- Transfection/methods
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Affiliation(s)
| | - Seksiri Arttamangkul
- Vollum Institute, Oregon Health & Science University, Portland, Oregon 97239, and
| | - Christopher J. Evans
- Department of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, Los Angeles, California 90024
| | - John T. Williams
- Vollum Institute, Oregon Health & Science University, Portland, Oregon 97239, and
| | - Mark von Zastrow
- Program in Neuroscience and
- Department of Psychiatry and Cellular and Molecular Pharmacology, University of California at San Francisco, San Francisco, California 94158
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Yamaza T, Kido MA, Wang B, Danjo A, Shimohira D, Murata N, Yoshinari M, Tanaka T. Distribution of substance P and neurokinin-1 receptors in the peri-implant epithelium around titanium dental implants in rats. Cell Tissue Res 2008; 335:407-15. [DOI: 10.1007/s00441-008-0720-7] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2008] [Accepted: 10/09/2008] [Indexed: 11/28/2022]
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Tang HB, Shiba E, Li YS, Morioka N, Zheng TX, Ogata N, Nakata Y. Involvement of voltage-gated sodium channel Na(v)1.8 in the regulation of the release and synthesis of substance P in adult mouse dorsal root ganglion neurons. J Pharmacol Sci 2008; 108:190-7. [PMID: 18845912 DOI: 10.1254/jphs.08163fp] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022] Open
Abstract
This study was conducted to determine whether Na(v)1.8 contributes to the release and/or synthesis of substance P (SP) in adult mice dorsal root ganglion (DRG) neurons. The SP released from cultured DRG neurons of Na(v)1.8 knock-out mice exposed to either capsaicin or KCl was significantly lower than that from wild-type (C57BL/6) mice based on a radioimmunoassay. The SP level of L6 DRG in Na(v)1.8 knock-out mice was also lower than that in wild-type mice. After chronic constriction injury (CCI) of the sciatic nerve, the level of SP decreased in the L6 ipsilateral DRG of wild-type but not Na(v)1.8 knock-out mice. The preprotachykinin-A (PPT-A) mRNAs in L4 - 6 DRGs of Na(v)1.8 knock-out mice also fell to half their normally abundant levels of expression. There were significant increases in Na(v)1.8 expression of the L6 contralateral DRG from wild-type mice and in the percentage of neurons expressing neurokinin-1 receptor in the cytosol of L6 DRGs from wild-type or Na(v)1.8 knock-out mice. These findings suggest that Na(v)1.8 is involved in the regulation of the release and synthesis of SP in the DRG neurons of wild-type mice.
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Affiliation(s)
- He-Bin Tang
- Department of Pharmacology, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima, Japan
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Abstract
Despite the large number of G-protein-coupled receptor (GPCR) types expressed in the CNS, little is known about their dynamics in neuronal cells. Dynamic properties of the somatostatin type 2A receptor were therefore examined in resting conditions and after agonist activation in living hippocampal neurons. Using fluorescence recovery after photobleaching experiments, we found that, in absence of ligand, the sst(2A) receptor is mobile and laterally and rapidly diffuse in neuronal membranes. We then observed by live-cell imaging that, after agonist activation, membrane-associated receptors induce the recruitment of beta-arrestin 1-enhanced green fluorescent protein (EGFP) and beta-arrestin 2-EGFP to the plasma membrane. In addition, beta-arrestin 1-EGFP translocate to the nucleus, suggesting that this protein could serve as a nuclear messenger for the sst(2A) receptor in neurons. Receptors are then recruited to preexisting clathrin coated pits, form clusters that internalize, fuse, and move to a perinuclear compartment that we identified as the trans-Golgi network (TGN), and recycle. Receptor cargoes are transported through a microtubule-dependent process directly from early endosomes/recycling endosomes to the TGN, bypassing the late endosomal compartment. Together, these results provide a comprehensive description of GPCR trafficking in living neurons and provide compelling evidence that GPCR cargoes can recycle through the TGN after endocytosis, a phenomenon that has not been anticipated from studies of non-neuronal cells.
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