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
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/31531 |
| Date | 09 May 2002 |
| Creators | Walthers, Eliza A. |
| Contributors | Moore, Frank L. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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