Identification of mesotocin and vasotocin nucleotide sequences in two species of urodele amphibian.

We amplified and identified, for the first time in urodele amphibians, cDNA sequences that encode preprovasotocin (preproVT) and prepromesotocin (preproMT) from two distinct urodelian species, Taricha granulosa (the rough-skinned newt) and Plethodon shermanii (the spotted salamander). Each of these cDNA sequences encoded proteins that contained the characteristics of known neurohypophysial peptide precursors; each sequence consisting of (1) a signal peptide, (2) VT- or MT-like peptides, (3) neurophysin, and for the preproVTs, (4) copeptin. In T. granulosa, cDNA sequences encoded for the nine amino acids that define VT or MT. In P. shermani, cDNA sequences encoded for the VT peptide and a previously unidentified isoform of MT, ([Val(4)]-MT).

Delta and mu opioid receptors from the brain of a urodele amphibian, the rough-skinned newt Taricha granulosa: cloning, heterologous expression, and pharmacological characterization.

Two full-length cDNAs, encoding delta (delta) and mu (mu) opioid receptors, were cloned from the brain of the rough-skinned newt Taricha granulosa, complementing previous work from our laboratory describing the cloning of newt brain kappa (kappa) and ORL1 opioid receptors. The newt delta receptor shares 82% amino acid sequence identity with a frog delta receptor and lower (68-70%) identity with orthologous receptors cloned from mammals and zebrafish. The newt mu receptor shares 79% sequence identity with a frog mu receptor, 72% identity with mammalian mu receptors, and 66-69% identity with mu receptors cloned from teleost fishes. Membranes isolated from COS-7 cells transiently expressing the newt delta receptor possessed a single, high-affinity (Kd = 2.4 nM) binding site for the nonselective opioid antagonist [3H]naloxone. In competition binding assays, the newt delta receptor displayed highest affinity for Met-enkephalin, relatively low affinity for Leu-enkephalin, beta-endorphin, and [D-penicillamine, D-penicillamine] enkephalin (DPDPE) (a delta-selective agonist in mammals), and very low affinity for mu-, kappa-, or ORL1-selective agonists. COS-7 cells expressing the newt mu receptor also possessed a high-affinity (Kd = 0.44 nM) naloxone-binding site that showed highest affinity for beta-endorphin, moderate-to-low affinity for Met-enkephalin and Leu-enkephalin and DAMGO (a mu-selective agonist in mammals), and very low affinity for DPDPE and kappa- or ORL1-selective agonists. COS-7 cells expressing either receptor type (delta or mu) showed very high affinity (Kd = 0.1-0.3 nM) for the nonselective opioid antagonist diprenorphine. Taricha granulosa expresses the same four subtypes (delta, mu, kappa, and ORL1) of opioid receptors found in other vertebrate classes, but ligand selectivity appears less stringent in the newt than has been documented in mammals.

Cloning proenkephalin from the brain of a urodele amphibian (Taricha granulosa) using a DOR-specific primer in a 3'RACE reaction.

A large cDNA fragment that codes for proenkephalin (PENK) was cloned from the rough-skinned newt, Taricha granulosa (GenBank Accession: AY817670). This 1299-bp PENK cDNA extends from the poly(A) sequence on the 3' end into the 5'-UTR (221bp) upstream of an open reading frame that codes for 264 amino acids and a stop codon. Within the precursor are five Met-enkephalin sequences and two C-terminally extended forms of Met-enkephalin (YGGFMRGV and YGGFMRY). The organization of the opioid core sequences within the newt PENK closely resembles that reported for other vertebrates. In this urodele amphibian, as in anurans, PENK does not contain the penultimate Leu-enkephalin opioid sequence found in mammals, and instead has in this position Met-enkephalin. PENK cDNA was amplified from newt brain in a RACE PCR targeting the 3' end of the newt delta opioid receptor (DOR). It remains to be determined whether generating the cDNA for the newt PENK while cloning its receptor was serendipitous or the result of a meaningful coincidence between the DOR and PENK sequences.

Cloning, heterologous expression and pharmacological characterization of a kappa opioid receptor from the brain of the rough-skinned newt, Taricha granulosa.

A full-length cDNA that encodes a kappa (kappa) opioid receptor has been isolated from the brain of a urodele amphibian, the rough-skinned newt Taricha granulosa. The deduced protein contains 385 amino acids and possesses features commonly attributed to G protein-coupled receptors, such as seven putative transmembrane domains. The newt kappa receptor has 75% sequence identity to kappa opioid receptors cloned from mammals, and 66% sequence identity to the kappa opioid receptor reported for the zebrafish, with the greatest divergence in the extracellular N-terminus, the second and third extracellular loops and the intracellular C-terminus. Membranes isolated from COS-7 cells expressing the newt kappa receptor possessed a single, high-affinity (Kd = 1.5 nM) binding site for the kappa-selective agonist U69593. In competition binding assays, the expressed newt kappa receptor displayed high affinity for the kappa-selective agonists GR89696, dynorphin A(1-13), U69593, U50488 and BRL52537, as well as the kappa-selective antagonist nor-binaltorphimine and the non-selective antagonist naloxone. Rank order potencies and affinity constants were similar in competition binding studies that used either whole brain homogenates or membranes isolated from COS-7 cells expressing the newt kappa receptor. The expressed receptor displayed essentially no affinity for the delta-selective agonist DPDPE ([d-penicillamine, d-penicillamine]enkephalin), but showed moderate affinity for the mu-selective agonist DAMGO ([d-Ala-MePhe, Gly-ol]enkephalin) and moderately high affinity for nociceptin (orphanin FQ), the endogenous ligand for the opioid receptor-like (ORL)1 receptor. These findings support the conclusions that a gene for the kappa opioid receptor is expressed in amphibians and that the pharmacology of the newt kappa receptor closely matches mammalian kappa opioid receptors. However, the functional dichotomy between the classic opioid receptors (kappa, delta, mu) and ORL1 appears less strict in amphibians than in mammals.

Neuroanatomical distribution of vasotocin and mesotocin in two urodele amphibians (Plethodon shermani and Taricha granulosa) based on in situ hybridization histochemistry.

Previous research suggests that considerable species-specific variation exists in the neuroanatomical distributions of arginine vasotocin (AVT) and mesotocin (MST), non-mammalian homologues of vasopressin and oxytocin. An earlier study in rough-skinned newts (Taricha granulosa) indicated that the neuroanatomical distribution of cells labeled for AVT-immunoreactivity (ir) was greater in this urodele amphibian than in any other species. It was unknown whether the widespread distribution of AVT-ir is unique to T. granulosa or a feature common among salamanders. Using in situ hybridization (ISH) histochemistry and gene-specific riboprobes, the current study labeled AVT and MST mRNA in T. granulosa and the red-legged salamander (Plethodon shermani). In T. granulosa, AVT ISH-labeled cells were found to be widespread and localized in brain areas including the dorsal and medial pallium, lateral and medial septum, bed nucleus of the stria terminalis, amygdala, preoptic area, ventral hypothalamus, nucleus isthmus, tectum mesencephali, inferior colliculus, and hindbrain. In P. shermani, the distribution of AVT ISH-labeled neurons matched that of T. granulosa, except in the lateral septum, ventral hypothalamus, and inferior colliculus, but did however include labeled cell bodies in the lateral pallium. The distribution of MST ISH-labeled cells was more restricted than AVT ISH labeling and was limited to regions of the preoptic area and ventral thalamus, which is consistent with the limited distribution of MST/OXY in other vertebrates. These findings support the conclusion that urodele amphibians possess a well-developed vasotocin system, perhaps more extensive than other vertebrate taxa.

Cloning, pharmacological characterization and tissue distribution of an ORL1 opioid receptor from an amphibian, the rough-skinned newt Taricha granulosa.

We have cloned and characterized an opioid receptor-like (ORL1; also referred to as NOP) receptor from a urodele amphibian, the rough-skinned newt Taricha granulosa The cDNA clone encodes a protein of 368 amino acids that contains the seven hydrophobic domains characteristic of G-protein-coupled receptors, and has the highest sequence identity to the frog (Rana pipiens) nociceptin-like and human ORL1 opioid receptors (79.6 and 68.4%, respectively). Saturation binding assays on membranes from COS-7 cells transiently expressing the newt ORL1 (nORL) receptor revealed a single, high-affinity (estimated Kd, 0.1974 nM) binding site for the ORL1-specific agonist [3H]orphanin FQ analog ([3H]oFQ). In competition binding assays, the [3H]oFQ-binding site, like the mammalian ORL1 receptor, had no affinity for the non-selective opioid receptor antagonist naloxone, the kappa-selective agonists U69593 and U50488, or the mu- and delta-selective opioid receptor agonists DAMGO and DPDPE, respectively. However, the nORL receptor displayed higher affinities for the kappa-selective agonists dynorphin A (1-13), dynorphin B, and dynorphin A (1-8) (Ki values, 2.8, 151.8, and 183.0 nM, respectively) than its mammalian homologue. The tissue distribution of the nORL receptor, as determined by reverse transcriptase PCR, was also found to differ from reports on the mammalian ORL1 receptor, with mRNA detected in brain, spinal cord, and lung, but not detected in a number of other peripheral tissues reported to express the receptor in mammals. This is the first report describing the expression and characterization of an amphibian ORL1 receptor, and contributes to our understanding of the evolution of the opioid system.