AuthorO'Day, Kenneth Bartels.
Committee ChairHampton, Jean
MetadataShow full item record
PublisherThe University of Arizona.
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.
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.