The neurohypophysial peptide system is involved in modulating a
variety of physiological, neurological, and behavioral responses in
vertebrates. The principal forms of these peptides in non-mammalian
tetrapods are vasotocin (VT) and mesotocin (MT). The studies described
in this thesis used pharmacological, molecular, and biochemical
techniques, along with phylogenetic analyses, to identify and characterize
the mRNA sequences encoding the neurohypophysial peptide precursor
proteins and their receptors in urodele amphibians.
The cDNAs encoding preproVT and preproMT were amplified by
PCR from the brains of two salamander species; the rough-skinned newt,
Taricha granulosa, and the red-legged salamander, Plethodon shermani.
The neurohypophysial peptides encoded by the identified Taricha cDNAs
were VT and MT; the Plethodon cDNAs encoded VT and a novel MT-like
peptide, [Valā“]-MT. Phylogenetic analyses grouped both the Taricha and
Plethodon preproVT and preproMT-like sequences with previously
identified tetrapod preproVT-like and preproMT-like sequences,
respectively. Additional analysis of the preproneurohypophysial sequences
indicated that gene conversion (non-homologous crossing over of DNA
sequences) appears to have occurred more frequently in mammals than in
other tetrapod classes.
The cDNAs encoding the VT receptor (VTR) and MT receptor (MTR)
were amplified from the brain of T. granulosa by PCR. Sequence identity,
and phylogenetic analysis, indicated that the Taricha MTR and VTR were
most similar to MTR/OTRs and V[subscript 1a]-like VTRs, respectively. Distribution of
PCR amplicons specific to the Taricha MTR and VTR matched previously
reported tissue distributions of MTRs and VTRs in other vertebrates in
every tissue but kidney, from which the Taricha primers were unable to
amplify a cDNA product. Binding experiments of transiently expressed
Taricha MTR indicated two binding states, and allowed the determination
of ligand binding affinities for this receptor. Inositol phosphate
accumulation assays demonstrated that the expressed Taricha MTR and
VTR cDNA produced functional receptors, and allowed calculation of ligand
potencies of activation and inhibition. Surprisingly, an antagonist
frequently used in behavioral experiments to specifically block VTR activity,
inhibited inositol phosphate accumulation in cells transfected with either the
Taricha MTR or VTR. In conclusion, these studies report the first identified cDNA
sequences encoding the preproVT, preproMT, MTR, and V[subscript 1a]-like VTR
proteins from urodele amphibians. / Graduation date: 2005
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/28695 |
| Date | 09 December 2004 |
| Creators | Searcy, Brian T. |
| Contributors | Moore, Frank L. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
Page generated in 0.0019 seconds