Graduate Thesis Or Dissertation
 

Identification and characterization of vasotocin and mesotocin peptides and receptors

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  • 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, [Val⁴]-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.
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