DOCSLIB.ORG

213393336.Pdf

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
213393336.Pdf The Development of Analytical Methods for Investigations of Dynorphin A 1-17 Metabolism in the Central Nervous System and Peripheral Tissues and Transport at the Blood Brain Barrier By Courtney D. Kuhnline Sloan B.S., Investigative and Medical Sciences and Chemistry, Saint Louis University, 2005 M.S., Pharmaceutical Chemistry, The University of Kansas, 2008 Submitted to the Department of Pharmaceutical Chemistry and the faculty of the Graduate School of the University of Kansas in partial fulfillment of the requirements for the degree of Doctor of Philosophy. ________________________________ Chairperson- Dr. Susan M. Lunte ________________________________ Dr. Kenneth L. Audus ________________________________ Dr. John F. Stobaugh ________________________________ Dr. Teruna J. Siahaan ________________________________ Dr. Jane V. Aldrich Dissertation Defense: February 11, 2011 The Dissertation Committee for Courtney Kuhnline Sloan certifies that this is the approved version of the following dissertation: The Development of Analytical Methods for Investigations of Dynorphin A 1-17 Metabolism, in the Central Nervous System and Peripheral Tissues, and Transport at the Blood Brain Barrier ________________________________ Chairperson- Dr. Susan M. Lunte Date approved:_______________________ ii This dissertation is dedicated to my parents, Pam and Mike Kuhnline, for always pushing me to do my absolute best at everything I have ever attempted, and to my husband, Patrick, for his continual encouragement and willingness to spend our first months as a married couple with me buried under my thesis. This work was possible because of the unconditional love and support I have been so fortunate to have from each of you. iii Abstract Dynorphin A 1-17 (Dyn A 1-17) is an endogenous neuropeptide that acts preferentially at the kappa opioid receptor. Elevated concentrations of Dyn A 1-17 have demonstrated neurotoxicity via a non-opioid mechanism, potentially mediated by NMDA (N-methyl-D- aspartate) receptors. This neurotoxicity has further been implicated in a variety of conditions including neuropathic pain, stress, depression, and neurological disorders including; Alzheimer’s and Parkinson’s disease. Unlike traditional small molecule neurotransmitters, peptides can undergo enzymatic degradation once released into the extracellular space and these metabolites can then go on to exhibit their own unique properties in vivo , often at sites far distal to their releasing cells. The investigation of dynorphin metabolism is especially crucial at blood brain barrier, where a peptide metabolite may exhibit substantially different transport properties. Traditional methods for the quantification of neuropeptides are immunoassays such as ELISA (enzyme linked immunosorbent assay) and RIA (radioimmunoassay). While these techniques have excellent limits of detection, cross-reactivity between related species is a significant issue. Therefore, it is essential to develop analytical methodologies capable of simultaneously determining the concentrations of both the parent peptide and its metabolites. In this thesis, the metabolism of Dyn A 1-17 in both the central nervous system and peripheral tissues as well as with an in vitro cell culture model of the blood brain barrier (BBB) is investigated. Methods for the separation and detection of Dyn A 1-17 and four of its key metabolites; Dyn A 1-6, Dyn A 1-8, Dyn A 1-13, and Dyn A 2-17 were developed using capillary electrophoresis (CE) with copper complexation and UV detection and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Dyn A 1-6 was found to be a major metabolite in the above mentioned systems and therefore its permeability at the BBB was iv investigated using bovine brain microvessel endothelial cells (BBMECs). The effect of this peptide on the permeability of the low molecular weight, low permeability compound fluorescein was also explored. In addition to CE and LC-MS/MS, microchip electrophoresis is also investigated. More specifically the development of an immunoaffinity microchip electrophoresis system was begun. Online immunoaffinity enables sample clean-up and preconcentration on the separation device. By combining immunoaffinity with highly efficient electrophoretic separations, an integrated device to investigate dynorphin metabolism is fabricated. Miniaturization decreases analysis time, significantly improving temporal resolution. Additionally preconcentration will improve limits of detection, making the detection of endogenous concentrations of dynorphin peptides from biological samples feasible. Towards this goal, antibody screening and some initial microchip electrophoresis studies with amperometric and laser-induced fluorescence detection were investigated. Future directions include the incorporation of this device with on-line microdialysis sampling and the development of an immunoaffinity microchip electrophoresis system for investigating neuropeptides implicated in neuropathic pain. v Acknowledgments I might be listed as the author on this thesis, but it was not a solitary effort by any means. There are many people without whom, this work would not have been possible and for all of you I am truly grateful. First, I would like to thank my advisor, Dr. Susan Lunte for allowing me to work in her lab during my time at KU. Thank you for listening to my interests and then assigning me a project that not only nurtured those interests but challenged me. You have given me more opportunities than I could ever have asked for while in graduate school. I am fairly certain my family thinks all I did was travel during my time here because of all of the conferences I was lucky enough to attend. Because of you I am a better scientist, teacher, and presenter. Thank you for always seeing the positive in my work, even if I didn’t see it at the time. I also must gratefully acknowledge Dr. Terry Phillips of the NIH for welcoming me into his lab for six months and teaching me a plethora of things about antibodies. My experience in your lab and in Washington, D.C. was an amazing opportunity and I truly appreciate all of your mentorship. I also must thank your lovely wife Jennifer for several wonderful dinners and your colleague Dr. Heather Kalish for all her assistance in the lab. Thanks to my committee members for taking time out of their busy schedules to be here today: Dr. Teruna Siahaan and Dr. Jane Aldrich and especially to my readers Dr. John Stobaugh and Dr. Ken Audus. A special note of thanks to people who have helped me in the lab or contributed to this project directly: Dr. Kelly Desino, Dr. Dan Mudra, and Dr. Rebecca Nofsinger for teaching me about mass spec and how to be a responsible user of a shared instrument; Glen Bisbee and Scott vi Toerber, amazingly talented engineers with the Waters corporation; Dr. Kshitij Patkar and Dr. Kelly Desino for teaching me the brain isolation procedures; Giuseppe and Dulan for later helping me with the isolation; Sara Thomas for the rat tissue harvests and the rest of Craig Lunte’s lab for letting me live my last year in the mass room in your lab; and the Forrest lab for letting me use your plate reader and occasionally your cell culture lab. Thank you to Nancy Helm for all of your help and friendship. Thanks to Ann and Karen for help with a variety of things, and Nicole for making my mass spec duties a little less stressful. Thank you to Gary Weber for help with posters, room reservations, and the always fun game of “Do you know where Sue is?” The graduate school experience would not be complete without the other people who surround you on a day-to-day basis in the lab. I am so lucky to have had such a wonderful “lab family”: Dr. Celeste Frankenfeld, Dr. Pradyot Nandi, Dr. David Fischer, Dr. Matthew Hulvey, Dr. Philip Livanec, Dhara Desai, Ryan Grigsby, Jessica Creamer, Anne Regel, Tom Linz, Dulan Gunasekara, and David Scott; undergraduates Emilie Mainz and Derek Jensen; and visiting scientists galore: Dr. Wendell Coltro, Dr. Mercedes Vasquez, Dr. Nobuyuki Suzuki (Nobusan), Dorothy Chrzaszcz, Jonas Bloedt, Emer Duffy, Christa Snyder, Giuseppe Caruso and Dr. Fracassi da Silva----- thank you all. I do not think I would have stayed sane without the help of Dr. Matt Hulvey. You are definitely the post doc extraordinaire and if I can be half as helpful to the students in the Bailey lab I will consider myself a success. Your friendship and support will never be forgotten. To Dave, Pradyot, and Ryan- thanks for letting me hang out when there were no girls in the lab and for always being willing to take a random lunch or dinner trip to find some new vii exotic (& of course spicy) food to try! Dave and Pradyot, thanks for always being up for a philosophical discussion about just about anything, especially one that eventually led us to the Phoggy Dog. Ryan, thank you for all of your help in the lab, when you leave the place may seriously fall apart. I owe a special thank you to the ladies of the Lunte Lab especially Dr. Celeste Frankenfeld, Dhara Desai, and Jessica Creamer for occupying the desk across from me and always being a sounding board for the “problem of the day”. Celeste, without you I may never have figured out how to get started in the lab. Dhara, you are truly the little sister I never had and I miss you! Thanks for always making me laugh. Jess, you made the end of grad school more fun that I thought it could be and for that I am so happy. Thanks to Jess and Anne for numerous coffee runs and trips out for great food. While in grad school I also had the pleasure of mentoring two undergraduate students: Dorothy Chrzaszcz and Emilie Mainz. Thank you for helping me hone my lab training and teaching skills and for always being so enthusiastic about research. You both served as wonderful reminders of how exciting and interesting research can be. All of you are amazing women and scientists and I am so happy to have worked with each of you.
Load more

Recommended publications
  • From Opiate Pharmacology to Opioid Peptide Physiology
    Upsala J Med Sci 105: 1-16,2000 From Opiate Pharmacology to Opioid Peptide Physiology Lars Terenius Experimental Alcohol and Drug Addiction Section, Department of Clinical Neuroscience, Karolinsku Institutet, S-I71 76 Stockholm, Sweden ABSTRACT This is a personal account of how studies of the pharmacology of opiates led to the discovery of a family of endogenous opioid peptides, also called endorphins. The unique pharmacological activity profile of opiates has an endogenous counterpart in the enkephalins and j3-endorphin, peptides which also are powerful analgesics and euphorigenic agents. The enkephalins not only act on the classic morphine (p-) receptor but also on the 6-receptor, which often co-exists with preceptors and mediates pain relief. Other members of the opioid peptide family are the dynor- phins, acting on the K-receptor earlier defined as precipitating unpleasant central nervous system (CNS) side effects in screening for opiate activity, A related peptide, nociceptin is not an opioid and acts on the separate NOR-receptor. Both dynorphins and nociceptin have modulatory effects on several CNS functions, including memory acquisition, stress and movement. In conclusion, a natural product, morphine and a large number of synthetic organic molecules, useful as drugs, have been found to probe a previously unknown physiologic system. This is a unique develop- ment not only in the neuropeptide field, but in physiology in general. INTRODUCTION Historical background Opiates are indispensible drugs in the pharmacologic armamentarium. No other drug family can relieve intense, deep pain and reduce suffering. Morphine, the prototypic opiate is an alkaloid extracted from the capsules of opium poppy.
    [Show full text]
  • Opposing Roles of Cotransmission of Dynorphin and Hypocretin on Reward and Motivation Xuan Li1, Nathan J
    COMMENTARY COMMENTARY Opposing roles of cotransmission of dynorphin and hypocretin on reward and motivation Xuan Li1, Nathan J. Marchant, and Yavin Shaham1 Behavioral Neuroscience Research Branch, Intramural Research Program, Department of A Brain stimulation Cocaine self- reward administration Health and Human Services, National Institute on Drug Abuse, National Institutes of Health, Baseline Impulsive behavior Baltimore, MD 21224 Hypocretin Dynorphin In PNAS, Muschamp et al. (1) report that sleep homeostasis (12), and—most relevant HCRTR1 antagonist: KOR signal dominates hypocretin and dynorphin are coexpressed in to the present study—the rewarding effects the same synaptic vesicles of hypothalamic of cocaine and other rewards via its action neurons. Their data from behavioral, phar- on VTA dopamine neurons (13, 14). macological, and electrophysiological studies In 2001, Chou et al. (15) reported that HCRTR1 antagonist + KOR antagonist: Balance restored suggest that hypocretin and dynorphin are hypocretin and dynorphin colocalize in the coreleased, and that they play opposing roles same hypothalamic neurons (15). In 2006, Li in cocaine self-administration, brain stimula- and van den Pol (16) extended these findings ’ tion reward, and impulsivity. The authors and provided electrophysiological data in- B Ventral tegmental area: data also suggest that the critical brain site for dicating that inhibitory dynorphin and ex- these opposing effects of coreleased hypocre- citatory hypocretin are simultaneously tin and dynorphin is the ventral tegmental coreleased after stimulation of hypothalamic area (VTA), the cell body region of the hypocretin neurons. The functional signifi- mesolimbic dopamine reward system (2). cance of hypocretin/dynorphin corelease is un- Fig. 1 provides a summary of the main find- known, because despite the above-mentioned ings from the study.
    [Show full text]
  • The Role of Protein Convertases in Bigdynorphin and Dynorphin a Metabolic Pathway
    Université de Montréal The Role of Protein Convertases in Bigdynorphin and Dynorphin A Metabolic Pathway par ALBERTO RUIZ ORDUNA Département de biomédecine vétérinaire Faculté de médecine vétérinaire Mémoire présenté à la Faculté de médecine vétérinaire en vue de l’obtention du grade de maître ès sciences (M.Sc.) en sciences vétérinaires option pharmacologie Décembre, 2015 © Alberto Ruiz Orduna, 2015 Résumé Les dynorphines sont des neuropeptides importants avec un rôle central dans la nociception et l’atténuation de la douleur. De nombreux mécanismes régulent les concentrations de dynorphine endogènes, y compris la protéolyse. Les Proprotéines convertases (PC) sont largement exprimées dans le système nerveux central et clivent spécifiquement le C-terminale de couple acides aminés basiques, ou un résidu basique unique. Le contrôle protéolytique des concentrations endogènes de Big Dynorphine (BDyn) et dynorphine A (Dyn A) a un effet important sur la perception de la douleur et le rôle de PC reste à être déterminée. L'objectif de cette étude était de décrypter le rôle de PC1 et PC2 dans le contrôle protéolytique de BDyn et Dyn A avec l'aide de fractions cellulaires de la moelle épinière de type sauvage (WT), PC1 -/+ et PC2 -/+ de souris et par la spectrométrie de masse. Nos résultats démontrent clairement que PC1 et PC2 sont impliquées dans la protéolyse de BDyn et Dyn A avec un rôle plus significatif pour PC1. Le traitement en C-terminal de BDyn génère des fragments peptidiques spécifiques incluant dynorphine 1-19, dynorphine 1-13, dynorphine 1-11 et dynorphine 1-7 et Dyn A génère les fragments dynorphine 1-13, dynorphine 1-11 et dynorphine 1-7.
    [Show full text]
  • The Role of Dynorphin in the Onset of Puberty in Female Lambs
    Graduate Theses, Dissertations, and Problem Reports 2015 The Role of Dynorphin in the Onset of Puberty in Female Lambs Justin Angelo Lopez Follow this and additional works at: https://researchrepository.wvu.edu/etd Recommended Citation Lopez, Justin Angelo, "The Role of Dynorphin in the Onset of Puberty in Female Lambs" (2015). Graduate Theses, Dissertations, and Problem Reports. 6110. https://researchrepository.wvu.edu/etd/6110 This Thesis is protected by copyright and/or related rights. It has been brought to you by the The Research Repository @ WVU with permission from the rights-holder(s). You are free to use this Thesis in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you must obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Thesis has been accepted for inclusion in WVU Graduate Theses, Dissertations, and Problem Reports collection by an authorized administrator of The Research Repository @ WVU. For more information, please contact [email protected]. The Role of Dynorphin in the Onset of Puberty in Female Lambs by Justin Angelo Lopez Thesis submitted to the Davis College of Agriculture, Natural Resources and Design at West Virginia University in partial fulfillment of the requirements for the degree of Master of Science in Reproductive Physiology Robert L. Goodman, Ph.D., Chair Stanley M. Hileman, Ph.D. Robert A. Dailey, Ph.D. Reproductive Physiology Morgantown, West Virginia 2015 Keywords: sheep, puberty, dynoprhin, kisspeptin, KNDy Copyright 2015 Justin Angelo Lopez ABSTRACT The Role of Dynorphin in the Onset of Puberty in Female Lambs Justin Angelo Lopez The neural mechanisms underlying the onset of puberty are not well understood.
    [Show full text]
  • Calcitonin Gene-Related Peptide and Other Peptides
    P1: KWW/KKL P2: KWW/HCN QC: KWW/FLX T1: KWW GRBT050-16 Olesen- 2057G GRBT050-Olesen-v6.cls July 9, 2005 4:30 ••Chapter 16 ◗ Calcitonin Gene-Related Peptide and Other Peptides Susan Brain and Lars Edvinsson Vasoactive peptides can be either stored or synthesized de THE CGRP FAMILY OF PEPTIDES novo before release from a range of tissues in the brain or from the walls of intracranial vasculature. In this chapter, The expression of mRNA from the calcitonin gene is tissue we concentrate on neuropeptides that are released from specific in that CGRP mRNA is predominantly expressed perivascular nerves. These include calcitonin gene-related in nerves and calcitonin mRNA in the thyroid (5). The 37 peptide (CGRP), substance P, neurokinin A, nociceptin, amino acid peptide CGRP belongs to a family that include somatostatin, and opioids (Table 16-1). The endothelium the more recently discovered peptides adrenomedullin produces the potent vasoconstrictors endothelin and an- that is primarily produced by non-neuronal tissues, espe- giotensin, and dilators such as nitric oxide, prostacyclin, cially vascular tissues and amylin that is mainly produced and endothelium-derived hyperpolarizing factors. In ad- in the pancreas. They share some structural homology (ap- dition there are circulating agents; among these the most proximately 25–40%) and also some, but not total, similar- potent is 5-hydroxytryptamine. The neuronal messengers ities in biological activities (see Brain and Grant [11] for stored in the intracranial vessels have been reviewed recent review). CGRP is abundant in the body and has a (32) and it was revealed that sympathetic nerves store wide distribution throughout the central and peripheral noradrenaline, neuropeptide Y, and ATP, the parasympa- nervous systems.
    [Show full text]
  • Five Decades of Research on Opioid Peptides: Current Knowledge and Unanswered Questions
    Molecular Pharmacology Fast Forward. Published on June 2, 2020 as DOI: 10.1124/mol.120.119388 This article has not been copyedited and formatted. The final version may differ from this version. File name: Opioid peptides v45 Date: 5/28/20 Review for Mol Pharm Special Issue celebrating 50 years of INRC Five decades of research on opioid peptides: Current knowledge and unanswered questions Lloyd D. Fricker1, Elyssa B. Margolis2, Ivone Gomes3, Lakshmi A. Devi3 1Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461, USA; E-mail: [email protected] 2Department of Neurology, UCSF Weill Institute for Neurosciences, 675 Nelson Rising Lane, San Francisco, CA 94143, USA; E-mail: [email protected] 3Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, Annenberg Downloaded from Building, One Gustave L. Levy Place, New York, NY 10029, USA; E-mail: [email protected] Running Title: Opioid peptides molpharm.aspetjournals.org Contact info for corresponding author(s): Lloyd Fricker, Ph.D. Department of Molecular Pharmacology Albert Einstein College of Medicine 1300 Morris Park Ave Bronx, NY 10461 Office: 718-430-4225 FAX: 718-430-8922 at ASPET Journals on October 1, 2021 Email: [email protected] Footnotes: The writing of the manuscript was funded in part by NIH grants DA008863 and NS026880 (to LAD) and AA026609 (to EBM). List of nonstandard abbreviations: ACTH Adrenocorticotrophic hormone AgRP Agouti-related peptide (AgRP) α-MSH Alpha-melanocyte stimulating hormone CART Cocaine- and amphetamine-regulated transcript CLIP Corticotropin-like intermediate lobe peptide DAMGO D-Ala2, N-MePhe4, Gly-ol]-enkephalin DOR Delta opioid receptor DPDPE [D-Pen2,D- Pen5]-enkephalin KOR Kappa opioid receptor MOR Mu opioid receptor PDYN Prodynorphin PENK Proenkephalin PET Positron-emission tomography PNOC Pronociceptin POMC Proopiomelanocortin 1 Molecular Pharmacology Fast Forward.
    [Show full text]
  • Calcitonin Gene-Related Peptide Regulates Expression of Neurokinin1 Receptors by Rat Spinal Neurons
    1816 • The Journal of Neuroscience, March 1, 2003 • 23(5):1816–1824 Calcitonin Gene-Related Peptide Regulates Expression of Neurokinin1 Receptors by Rat Spinal Neurons Virginia S. Seybold,1 Kenneth E. McCarson,2 Paul G. Mermelstein,1 Rachel D. Groth,1 and Lia G. Abrahams1 1Department of Neuroscience, University of Minnesota, Minneapolis, Minnesota 55455, and 2Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas 66160 Although neurokinin 1 (NK1) receptors contribute to hyperalgesia, and their expression is increased in the spinal cord during peripheral inflammation, little is known regarding the signaling molecules and the second messenger pathways that they activate in regulating the expression of the NK1 receptor gene. Because the promoter region of the NK1 receptor contains a cAMP response element (CRE), we tested the hypothesis that calcitonin gene-related peptide (CGRP) regulates the expression of NK1 receptors via a pathway involving activation of the transcription factor cAMP response element binding protein (CREB). Experiments were conducted on primary cultures of neonatal rat spinal neurons. Treatment of cultures with CGRP for 8–24 hr increased 125I-substance P binding on spinal neurons; the increase in binding was preceded by an elevation in NK1 receptor mRNA. The CGRP-induced change in 125I-substance P binding was concentration-dependent and was inhibited by the antagonist CGRP8–37. CGRP increased phosphorylated CREB immunoreactivity and CRE-dependent transcription in neurons, indicating the involvement of the transcription factor CREB. Evidence that CGRP increased cAMP levels in spinal neurons and that the protein kinase A inhibitor H89 attenuated CGRP-induced CRE-dependent transcription suggests that the intracellular pathway stimulated by CGRP leads to activation of protein kinase A.
    [Show full text]
  • Distribution of Prodynorphin Mrna and Its Interaction with the NPY System in the Mouse Brain Neuropeptides 40 (2): 115-123, 2006
    Lin et al.: Distribution of prodynorphin mRNA and its interaction with the NPY system in the mouse brain Neuropeptides 40 (2): 115-123, 2006 Distribution of prodynorphin mRNA and its interaction with the NPY system in the mouse brain Shu Lina, Dana Boeya, Nicola Leea, Christoph Schwarzerb, Amanda Sainsburya, Herbert Herzoga, a Neurobiology Program, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australiab Department of Pharmacology, Medical University of Innsbruck, Peter- Mayr-Strasse 1a, 6020 Innsbruck, Austria Abstract Using radioactive in situ hybridisation, the distribution of prodynorphin mRNA in the brains of C57Bl/6 mice was systemically investigated, and double-labelling in situ hybridisation was used to determine the extent to which neuropeptide Y (NPY) and prodynorphin mRNAs were co-expressed. Our results demonstrate that prodynorphin mRNA expression in the mouse brain is localised at specific subregions of the olfactory bulb, cortex, hippocampus, amygdala, basal ganglia, thalamus, hypothalamus, mesencephalon and myelencephalon. Among the regions displaying the most intense labelling were the olfactory tubercle, lateral septum (LS), caudate putamen (Cpu), central amygdaloid nucleus (Ce), paraventricular hypothalamic nucleus (PVN), supraoptic nucleus (SO), lateral hypothalamic area (LHA), ventromedial hypothalamic nucleus (VMH), lateral reticular nucleus (LRt) and solitary tract nucleus (NTS). In the arcuate nucleus of the hypothalamus (Arc), double- labelling in situ hybridisation revealed that prodynorphin expressing neurons also contained NPY mRNA, with a co-localisation rate of approximately 88% in the lateral part of the Arc, and 79% in the dorsal part of the Arc, respectively, suggesting potential overlapping functions of these two neurotransmitters in feeding type behaviour.
    [Show full text]
  • Dynorphin- and Enkephalin-Like Lmmunoreactivity Is Altered in Limbic-Basal Ganglia Regions of Rat Brain After Repeated Electroconvulsive Shock’
    The Journal of Neuroscience March 1966, 6(3): 644-649 Dynorphin- and Enkephalin-like lmmunoreactivity is Altered in Limbic-Basal Ganglia Regions of Rat Brain After Repeated Electroconvulsive Shock’ T. Kanamatsu, J. F. McGinty,* C. L. Mitchell, and J. S. Hong Laboratory of Behavioral and Neurological Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, and *Department of Anatomy, School of Medicine, East Carolina University, Greenville, North Carolina 27834 In an attempt to determine whether the opioid peptides derived thalamus, whereas the level of hypothalamic /3-endorphin is from prodynorphin participate in the effects of electroconvulsive unaltered (Hong et al., 1979). Our recent study, using in vitro shock (ECS), we used radioimmunoassay and immunocyto- cell free translation or blot hybridization with a rat preproen- chemistry to measure dynorphin-like immunoreactivity (DN-LI) kephalin A cDNA clone to estimate the level of mRNA coding in various rat brain regions after repeated ECS treatments. Ten for preproenkephalin A, suggestedthat the increasein hypo- daily ECSs caused a significant increase in dynorphin A (1-8)- thalamic ME-L1 after repeated ECS is due to an increase in LI in most limbic-basal ganglia structures, including hypothal- biosynthesis(Yoshikawa et al., 1985). These observations raise amus (50%), striatum (30%), and septum (30%). No significant the possibility that an increasein enkephalinergicneuronal ac- change was found in the frontal cortex or the neurointermediate tivity is related to ECS-elicited phenomena. lobe of the pituitary. In contrast, 10 ECS treatments depleted It was recently reported that the level of hippocampal dy- DN-LI in hippocampal mossy fibers by 64%.
    [Show full text]
  • Mammalian Neuropeptides As Modulators of Microbial Infections: Their Dual Role in Defense Versus Virulence and Pathogenesis
    International Journal of Molecular Sciences Review Mammalian Neuropeptides as Modulators of Microbial Infections: Their Dual Role in Defense versus Virulence and Pathogenesis Daria Augustyniak 1,* , Eliza Kramarska 1,2, Paweł Mackiewicz 3, Magdalena Orczyk-Pawiłowicz 4 and Fionnuala T. Lundy 5 1 Department of Pathogen Biology and Immunology, Faculty of Biology, University of Wroclaw, 51-148 Wroclaw, Poland; [email protected] 2 Institute of Biostructures and Bioimaging, Consiglio Nazionale delle Ricerche, 80134 Napoli, Italy 3 Department of Bioinformatics and Genomics, Faculty of Biotechnology, University of Wroclaw, 50-383 Wroclaw, Poland; pamac@smorfland.uni.wroc.pl 4 Department of Chemistry and Immunochemistry, Wroclaw Medical University, 50-369 Wroclaw, Poland; [email protected] 5 Wellcome-Wolfson Institute for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, Belfast BT9 7BL, UK; [email protected] * Correspondence: [email protected]; Tel.: +48-71-375-6296 Abstract: The regulation of infection and inflammation by a variety of host peptides may represent an evolutionary failsafe in terms of functional degeneracy and it emphasizes the significance of host defense in survival. Neuropeptides have been demonstrated to have similar antimicrobial activities to conventional antimicrobial peptides with broad-spectrum action against a variety of microorganisms. Citation: Augustyniak, D.; Neuropeptides display indirect anti-infective capacity via
    [Show full text]
  • Big Dynorphin, a Prodynorphin-Derived Peptide Produces NMDA Receptor-Mediated Effects on Memory, Anxiolytic-Like and Locomotor Behavior in Mice
    Neuropsychopharmacology (2006) 31, 1928–1937 & 2006 Nature Publishing Group All rights reserved 0893-133X/06 $30.00 www.neuropsychopharmacology.org Big Dynorphin, a Prodynorphin-Derived Peptide Produces NMDA Receptor-Mediated Effects on Memory, Anxiolytic-Like and Locomotor Behavior in Mice ,1,2 1 2 1 2 Alexander Kuzmin* , Nather Madjid , Lars Terenius , Sven Ove Ogren and Georgy Bakalkin 1Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden; 2Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden Effects of big dynorphin (Big Dyn), a prodynorphin-derived peptide consisting of dynorphin A (Dyn A) and dynorphin B (Dyn B) on memory function, anxiety, and locomotor activity were studied in mice and compared to those of Dyn A and Dyn B. All peptides administered i.c.v. increased step-through latency in the passive avoidance test with the maximum effective doses of 2.5, 0.005, and 0.7 nmol/animal, respectively. Effects of Big Dyn were inhibited by MK 801 (0.1 mg/kg), an NMDA ion-channel blocker whereas those of dynorphins A and B were blocked by the k-opioid antagonist nor-binaltorphimine (6 mg/kg). Big Dyn (2.5 nmol) enhanced locomotor activity in the open field test and induced anxiolytic-like behavior both effects blocked by MK 801. No changes in locomotor activity and no signs of anxiolytic-like behavior were produced by dynorphins A and B. Big Dyn (2.5 nmol) increased time spent in the open branches of the elevated plus maze apparatus with no changes in general locomotion. Whereas dynorphins A and B (i.c.v., 0.05 and 7 nmol/animal, respectively) produced analgesia in the hot-plate test Big Dyn did not.
    [Show full text]
  • Specific Regions of the Rat Brain
    Proc. Nad. Acad. Sci. USA Vol. 81, pp. 6886-6889, November 1984 Neurobiology Differential processing of prodynorphin and proenkephalin in specific regions of the rat brain (dynorphins/neo-endorphins/[Leulenkephalin/[Metlenkephalin-Arg6-Gly7_Leu'/rat brain nuclei) NADAV ZAMIR*, ECKARD WEBERt, MIKLOS PALKOVITS*, AND MICHAEL BROWNSTEIN* *Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, MD 20205; and tNancy Pritzker Laboratory of Behavioral Neurochemistry, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA 94305 Communicated by Seymour S. Kety, July 10, 1984 ABSTRACT Prodynorphin-derived peptides [dynorphin technique, from 300-,um thick frozen coronal sections cut in A (Dyn A)-(1-17), Dyn A-(1-8), Dyn B, a-neo-endorphin, and a cryostat at -10°C (21). Tissue samples were placed in Ep- (3-neo-endorphin] and proenkephalin-derived peptides pendorf tubes containing 200 M1 of0.1 M HC1 and transferred {[Leu]enkephalin ([Leu]Enk) and [Metlenkephalin-Arg6-Gly7- to a boiling water bath for 10 min. Samples were chilled in Leu8 ([Met]Enk-Arg-Gly-Leu)} in selected brain areas of the ice and then homogenized by sonication, and 20-,u aliquots rat were measured by specific radioimmunoassays. We report of the homogenates were removed for protein determination here that different regions of rat brain contain strikingly dif- (22). The extracts were centrifuged at 2000 x g for 10 min at ferent proportions of the prodynorphin and proenkephalin-de- 4°C. The supernatants were transferred to 12 x 75 mm poly- rived peptides. There is a molar excess of a-neo-endorphin- propylene or polystyrene tubes (the latter for RIAs of derived peptides over Dyn B and Dyn A-derived peptides in [Leu]Enk and [Met]Enk-Arg-Gly-Leu) and evaporated to many brain areas.
    [Show full text]