Graduate Thesis Or Dissertation
 

An opioid-like receptor in the roughskin newt, Taricha granulosa

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  • 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.
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file details WalthersElizaA2003.pdf 2017-08-08 Pubblico Scaricare