Localization of alpha-2 adrenergic receptor subtypes using subtype-specific antibodies.

Persistent Link:
http://hdl.handle.net/10150/187223
Title:
Localization of alpha-2 adrenergic receptor subtypes using subtype-specific antibodies.
Author:
Huang, Yi.
Issue Date:
1995
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Three subtypes of alpha-2 adrenergic receptors (α₂AR) have been identified: α₂A, α₂B and α₂C. α₂ARs are known to mediate a number of functions in many tissues. It is not clear, however, what the distribution of α₂AR subtypes is in these tissues. The distribution of α₂AR subtypes is fundamental to understanding receptor function and the development of more effective and specific α₂-adrenergic agents for therapeutic use. The working hypothesis for this dissertation is that there are specific subtypes of α₂ARs in tissues and in cells that have discrete localizations and may subserve different physiological function. To test this hypothesis two specific aims have been raised. The first aim was: the generation of subtype-specific antibodies. Experiments were conducted to express fusion proteins containing part of the third intracellular loop of each α₂AR. Chickens were immunized with fusion proteins and antibodies were purified from the yolk of the chicken eggs. The antibodies have complete subtype specificity as characterized by immunofluorescence studies on COS-7 cells transfected with DNA encoding the α₂ARs. The second aim was: the immunohistochemical localization of α₂AR subtypes in the primary culture of rat spinal cord, anterior segments of human and rabbit eyes and transfected COS-7 cells. Experiments were conducted to localize the α₂AR subtypes in cultured cells and tissues using indirect immunofluorescence techniques. The immunofluorescence results were confirmed by alternative approaches, e.g. reverse transcription-PCR, ligand binding and cAMP assay. The experimental results showed that multiple α₂AR subtypes are expressed in one tissue and that specific subtypes of α₂ARs are expressed in different tissues. Using a dual-labeling technique, two subtypes were co-localized in the same neuronal cell of the rat spinal cord. Furthermore, the immunofluorescence studies on the transfected COS-7 cells showed that the three subtypes of α₂ARs displayed different subcellular localization. Taken together the results of the studies presented in this dissertation provide evidence in support of the working hypothesis. The present work has provided a new opening in the study of the localization of α₂AR subtypes. This information provides new insights into the understanding of α₂AR functions in tissues.
Type:
text; Dissertation-Reproduction (electronic)
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Physiological Sciences; Graduate College
Degree Grantor:
University of Arizona
Committee Chair:
Regan, John W.

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleLocalization of alpha-2 adrenergic receptor subtypes using subtype-specific antibodies.en_US
dc.creatorHuang, Yi.en_US
dc.contributor.authorHuang, Yi.en_US
dc.date.issued1995en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractThree subtypes of alpha-2 adrenergic receptors (α₂AR) have been identified: α₂A, α₂B and α₂C. α₂ARs are known to mediate a number of functions in many tissues. It is not clear, however, what the distribution of α₂AR subtypes is in these tissues. The distribution of α₂AR subtypes is fundamental to understanding receptor function and the development of more effective and specific α₂-adrenergic agents for therapeutic use. The working hypothesis for this dissertation is that there are specific subtypes of α₂ARs in tissues and in cells that have discrete localizations and may subserve different physiological function. To test this hypothesis two specific aims have been raised. The first aim was: the generation of subtype-specific antibodies. Experiments were conducted to express fusion proteins containing part of the third intracellular loop of each α₂AR. Chickens were immunized with fusion proteins and antibodies were purified from the yolk of the chicken eggs. The antibodies have complete subtype specificity as characterized by immunofluorescence studies on COS-7 cells transfected with DNA encoding the α₂ARs. The second aim was: the immunohistochemical localization of α₂AR subtypes in the primary culture of rat spinal cord, anterior segments of human and rabbit eyes and transfected COS-7 cells. Experiments were conducted to localize the α₂AR subtypes in cultured cells and tissues using indirect immunofluorescence techniques. The immunofluorescence results were confirmed by alternative approaches, e.g. reverse transcription-PCR, ligand binding and cAMP assay. The experimental results showed that multiple α₂AR subtypes are expressed in one tissue and that specific subtypes of α₂ARs are expressed in different tissues. Using a dual-labeling technique, two subtypes were co-localized in the same neuronal cell of the rat spinal cord. Furthermore, the immunofluorescence studies on the transfected COS-7 cells showed that the three subtypes of α₂ARs displayed different subcellular localization. Taken together the results of the studies presented in this dissertation provide evidence in support of the working hypothesis. The present work has provided a new opening in the study of the localization of α₂AR subtypes. This information provides new insights into the understanding of α₂AR functions in tissues.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplinePhysiological Sciencesen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.chairRegan, John W.en_US
dc.contributor.committeememberHoyer, Patricia B.en_US
dc.contributor.committeememberLai, Josephine Y.en_US
dc.contributor.committeememberLynch, Ronald M.en_US
dc.contributor.committeememberRoeske, William R.en_US
dc.identifier.proquest9603371en_US
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