Thesis (Ph. D.)--University of Hawaii at Manoa, 1994. / Includes bibliographical references (leaves 85-101). / Microfiche. / xvi, 101 leaves, bound ill. 29 cm

No description available.

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

This report describes the synthesis, chemical derivation, and pharmacological and behavioral characterization of several unique classes of opioid receptor modulators. In chapter one, a general overview of opioid receptor history, signaling biology, and therapeutic applications is provided. Also reviewed are several topics of high current interest, including, biased signaling, opioid receptor splice variants/heteromers, and applications of opioid modulators in the treatment of mood disorders. This introduction aims to frame the work that follows, and emphasize to the reader the untapped potential of the opioid receptor system, particularly in the realm of therapeutics development. Chapter two discusses the development of several new C-H activation reactions to provide rapid access to the core molecular scaffold of alkaloids from Tabernanthe iboga. The methods described permit the expedient construction of structurally diverse ibogamine analogs via a modular approach. In chapter three, this work is extended by applying the new reaction methodologies to explore a novel class of oxaibogamine analogs, which act as opioid receptor agonists and antagonists. The thorough exploration of structure-activity relationships within this skeleton is described, along with the pharmacological characterization of several select analogs as biased agonists at both the kappa- and mu-opioid receptors. This section concludes with a discussion of potential therapeutic applications for the synthesized compounds as new analgesics and antidepressants, and future goals and plans for this structural class. In chapter four, the isolation and pharmacological study of several alkaloids of Mitragyna speciosa is presented. Mitragynine, the primary natural alkaloid in this plant, is isolated, along with several naturally occurring analogs, and the modulatory activity of these compounds at the opioid receptors is fully characterized. Further, preliminary results are presented suggesting activity of these alkaloids at several other classes of central nervous system targets, including serotonin and adrenergic receptors. Also discussed are the preparations of semi-synthetic and fully synthetic mitragynine derivatives, including a total synthesis of mitragynine itself. These novel analogs are applied to explore key structure-activity relationships in this unusual opioid-active scaffold. Again, potential applications of Mitragyna alkaloid analogs in the treatment of pain and depression are discussed. In the final chapter, I describe our discovery that tianeptine, a clinically used atypical antidepressant of previously unknown mechanism of action, acts as an agonist of both the mu- and delta-opioid receptors. Activation of the mu-opioid receptor is thus proposed as the initial molecular-level event responsible for eliciting the beneficial therapeutic effects of this agent. This hypothesis is integrated with the large body of literature describing this compound, and mechanistic theories connecting the opioid activity of tianeptine to previous observations are described, with a particular emphasis on indirect modulation of glutamate signaling. Behavioral studies in mice employing both genetic knockout and pharmacological inhibition are then used to confirm the involvement of the opioid receptors in tianeptine's mechanism of action. Also described are thorough explorations of opioid structure-activity relationships within the tianeptine scaffold, and the design and synthesis of novel analogs having improved pharmacokinetic properties. It is hoped that these derivatives may one day serve as new therapeutic options for patients suffering from treatment-resistant depression.

The main objectives of the current study were to obtain the complete cDNA sequence of an opioid-like receptor from an amphibian, the roughskin newt, Taricha granulosa, and investigate the receptor's tissue distribution and regulation following chronic exposure to the glucocorticoid corticosterone (CORT). Degenerate primers designed in highly conserved regions of characterized opioid receptors were used to amplify opioid-like receptor fragments from a newt brain cDNA library. Receptor fragments with high sequence identity to the orphanin opioid receptor type, also termed the 'opioid receptor-like' (ORL1) receptor, were selected for 3' and 5' RACE (rapid amplification of cDNA ends) reactions to obtain the full-length receptor cDNA sequence. By this approach, we obtained a cDNA sequence that putatively encodes a 368 amino acid protein with high sequence identity (57%) to the human ORL1 receptor. Therefore, hereafter we refer to this receptor as the newt ORL1-like (nORL) receptor. The nORL receptor also has identity with the mammalian kappa (K) opioid receptor at a number of residues that may enable it to recognize both ORL1- and K- receptor selective ligands. The tissue distribution of the nORL receptor was determined by reverse-transcriptase polymerase chain reaction (PCR). RNA from a variety of tissues was reverse-transcribed into cDNA using an oligo-dT primer, and the resultant cDNA was used as template in PCR reactions with nORL receptor-specific primers. Appropriately sized amplicons were produced in reactions with cDNA template originating from newt brain, spinal cord, and lungs. No amplification occurred in reactions with template cDNA from newt spleen, small intestine, heart, liver, sperm duct, bladder, or kidney. The regulation of the nORL receptor following chronic exposure to the glucocorticoid corticosterone was investigated using real-time PCR. Animals were exposed continuously to CORT for 10 days using surgically implanted Silastic capsules packed with CORT powder. Control animals received empty Silastic capsules, or no treatment. The relative quantitation of the nORL receptor messenger RNA (mRNA) was achieved by real-time PCR, and mRNA levels for the hormone-treated animals were compared to those of the controls. The same samples were used for the relative quantitation of intracellular glucocorticoid receptor (iGR) mRNA. There was no change in the expression of mRNA for the nORL receptor or the iGR following chronic exposure to CORT as compared to the controls. In conclusion, this study provides evidence for an opioid-like receptor in the roughskin newt that has high sequence identity to the mammalian ORL1 opioid receptor. To the best of our knowledge, this is the first complete opioid receptor cDNA sequence obtained for an amphibian. The nORL receptor appears to principally function in central nervous system (CNS) processes in the newt, as evidenced by its primary localization to brain and spinal cord. The role of the nORL receptor in the periphery may be limited to a function in the lungs, and awaits further investigation. The current study was also the first to investigate the effects of a stress hormone on the regulation of an opioid receptor in an amphibian. Our results indicate that chronic exposure to the stress hormone corticosterone does not impact the levels of nORL receptor or intracellular glucocorticoid receptor mRNA in the newt spinal cord. / Graduation date: 2003

published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

The Mu Opioid Receptor (MOR) is a G protein-coupled receptor (GPCR) important for pain regulation. Opioid agonists have long been the most effective treatment for most types of pain; however, this class of drugs is highly problematic due to the combination of several dangerous side effects like addiction, tolerance, and respiratory depression. Recently, a dramatic rise in opioid prescriptions has led to a nationwide opioid epidemic. Efforts to develop novel opioids with improved therapeutic profiles have led to work suggesting that MOR signaling through G proteins leads to analgesia while signaling through arrestin leads to respiratory depression and tolerance. However, more recent work has raised questions about which aspects of arrestin signaling and function contribute to these side effects. Additionally, the overall complexity of arrestin function especially with regard to trafficking at the cell membrane has recently come in to clearer view. Here, we use single-molecule tracking to describe membrane diffusion behavior of single MORs before and after agonist treatment in heterologous cells. By tracking individual MORs, we have revealed cell-context specific rules for MOR immobilization and endocytosis and shown that these processes depend on receptor density as well as the local availability of arrestin molecules.