Rough-skinned Newt Taricha Granulosa Research Articles

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 (δ) and mu (μ) 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 (κ) and ORL1 opioid receptors. The newt δ receptor shares 82% amino acid sequence identity with a frog δ receptor and lower (68–70%) identity with orthologous receptors cloned from mammals and zebrafish. The newt μ receptor shares 79% sequence identity with a frog μ receptor, 72% identity with mammalian μ receptors, and 66–69% identity with μ receptors cloned from teleost fishes. Membranes isolated from COS-7 cells transiently expressing the newt δ receptor possessed a single, high-affinity ( K d = 2.4 nM) binding site for the nonselective opioid antagonist [ 3H]naloxone. In competition binding assays, the newt δ receptor displayed highest affinity for Met-enkephalin, relatively low affinity for Leu-enkephalin, β-endorphin, and [ d-penicillamine, d-penicillamine] enkephalin (DPDPE) (a δ-selective agonist in mammals), and very low affinity for μ-, κ-, or ORL1-selective agonists. COS-7 cells expressing the newt μ receptor also possessed a high-affinity ( K d = 0.44 nM) naloxone-binding site that showed highest affinity for β-endorphin, moderate-to-low affinity for Met-enkephalin and Leu-enkephalin and DAMGO (a μ-selective agonist in mammals), and very low affinity for DPDPE and κ- or ORL1-selective agonists. COS-7 cells expressing either receptor type (δ or μ) showed very high affinity ( K d = 0.1–0.3 nM) for the nonselective opioid antagonist diprenorphine. Taricha granulosa expresses the same four subtypes (δ, μ, κ, 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, 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.

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.

COEVOLUTION OF DEADLY TOXINS AND PREDATOR RESISTANCE: SELF-ASSESSMENT OF RESISTANCE BY GARTER SNAKES LEADS TO BEHAVIORAL REJECTION OF TOXIC NEWT PREY

Deadly toxins and resistance to them are an evolutionary enigma. Selection for increased resistance does not occur if predators do not survive encounters with toxic prey. Similarly, deadly toxins are of no advantage to individual prey if it dies delivering the toxins. For individual selection to drive the coevolutionary arms race between resistant predators and lethal prey, the survivorship of individual predators must covary with their resistance. The extreme toxicity of the rough skinned newt Taricha granulosa appears to have coevolved with resistance in its predator, the common garter snake Thamnophis sirtalis, yet the mechanism by which individual selection can operate has been unclear in this and other lethal prey-predator systems. We show that individual snakes assess their own resistance relative to newt toxicity and reject prey too toxic to consume. Rejected newts all survived attacks and attempted ingestion by snakes that sometimes lasted over 50 min. Behavioral moderation of toxin exposure by snakes provides the association between individual resistance and fitness necessary for coevolution of lethal toxins and resistance to occur.

Metabolic and Biochemical Correlates of Thermal Acclimation in the Rough-Skinned Newt Taricha granulosa

Taricha granulosa (the rough-skinned newt) exhibited no thermal acclimation of metabolism when measured at a low experimental temperature (ET) (5 C) but underwent pronounced acclimation at a high ET (20 C). Thermal acclimation markedly altered the substrate concentration at which enzyme activity was 50% of maximum activity ($K_{s}$) for two enzymes, lactate dehydrogenase (LDH) and malate dehydrogenase (MDH). In general, the $K_{s}$ decreased after acclimation to low temperatures. Maximum activity of both enzymes, by contrast, was unaffected by thermal acclimation. No qualitative differences in isozymes from warm-and cold-acclimated salamanders were evident from starch gel electrophoresis of LDH, MDH, or IDH (isocitrate dehydrogenase). Acclimation to cold temperatures had no effect on the proportions of most phospholipids in the liver but increased the proportions of unsaturated fatty acids and decreased sphingomyelin content. In most respects, Taricha granulosa resembled other ectothermic vertebrates in i...

Migration, overwintering, and reproductive patterns of Taricha granulosa on southern Vancouver Island.

Sexual dichotomy in habitat selection during the nonbreeding season was studied in the rough-skinned newt Taricha granulosa from November 1970 to August 1973 on southern Vancouver Island. Field results show that in this area adult females normally migrate from breeding ponds to overwinter on land. Adult males normally remain permanently aquatic. Related observations on the reproductive cycle of T. granulosa show that females, who lay their eggs singly, deposit them for a period of a few days, followed by a period of nonegglaying. This process is repeated at intervals. Oviposition can occur from late April to July in this area. Females who were induced to ovulate in the laboratory each deposited three eggs per day for 5 days before opposition ceased. Thus the reproductive cycles of field and taboratory-acclimated females are correlated.

The Rate of Metabolic Temperature Acclimation in the Frog, Rana temporaria

Previous articleNext article No AccessThe Rate of Metabolic Temperature Acclimation in the Frog, Rana temporariaMikko N. E. HarriMikko N. E. Harri Search for more articles by this author PDFPDF PLUS Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by Volume 46, Number 2Apr., 1973 Article DOIhttps://doi.org/10.1086/physzool.46.2.30155595 Views: 9Total views on this site Citations: 19Citations are reported from Crossref Journal History This article was published in Physiological Zoology (1928-1998), which is continued by Physiological and Biochemical Zoology (1999-present). PDF download Crossref reports the following articles citing this article:E S Savelieva INVESTIGATION OF INSULIN-, GLUCAGON-, SOMATOSTATIN-POSITIV CELLS LOCALIZATION IN THE LANGERHANS’ ISLENDS IN DIFFERENT PERIODS OF BIOCYCLE OF ANURA, I.P. Pavlov Russian Medical Biological Herald 21, no.11 (Dec 2013): 7–10.https://doi.org/10.17816/PAVLOVJ201317-10Ari J Tiiska, Kari Y.H Lagerspetz Effects of thermal acclimation on nervous conduction and muscle contraction in the frog Rana temporaria, Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 124, no.33 (Nov 1999): 335–342.https://doi.org/10.1016/S1095-6433(99)00125-7Donald G Dunlap The effects of differing photoperiods during thermal acclimation on oxygen consumption in the hylid frogs, Acris crepitans and Pseudacris triseriata, Comparative Biochemistry and Physiology Part A: Physiology 94, no.33 (Jan 1989): 519–523.https://doi.org/10.1016/0300-9629(89)90132-1Loren U. Williamson, James R. Spotila, Edward A. Standora Growth selected temperature and CTM of young snapping turtles, Chelydra serpentina, Journal of Thermal Biology 14, no.11 (Jan 1989): 33–39.https://doi.org/10.1016/0306-4565(89)90027-2E.Don Stevens Acclimation temperature markedly alters the motivation to feed in the toad, Bufo americanus, Journal of Thermal Biology 13, no.22 (Apr 1988): 73–78.https://doi.org/10.1016/0306-4565(88)90015-0E. Don Stevens The effect of acclimation temperature on feeding performance in the toad, Bufo americanus, Comparative Biochemistry and Physiology Part A: Physiology 89, no.22 (Jan 1988): 131–135.https://doi.org/10.1016/0300-9629(88)91069-9Martin E. Feder Acclimation to constant and variable temperatures in plethodontid salamanders—I. Rates of oxygen consumption, Comparative Biochemistry and Physiology Part A: Physiology 81, no.33 (Jan 1985): 673–682.https://doi.org/10.1016/0300-9629(85)91046-1Martin E. Feder, Allen G. Gibbs, Gerard A. Griffith, Joyce Tsuji Thermal acclimation of metabolism in salamanders: Fact or artefact?, Journal of Thermal Biology 9, no.44 (Oct 1984): 255–260.https://doi.org/10.1016/0306-4565(84)90005-6 Martin E. Feder Metabolic and Biochemical Correlates of Thermal Acclimation in the Rough-Skinned Newt Taricha granulosa, Physiological Zoology 56, no.44 (Sep 2015): 513–521.https://doi.org/10.1086/physzool.56.4.30155874G.D. Parry The influence of the cost of growth on ectotherm metabolism, Journal of Theoretical Biology 101, no.33 (Apr 1983): 453–477.https://doi.org/10.1016/0022-5193(83)90150-9Katsuji Tsugawa Effects of cold acclimation on the standard metabolic rate and osmotic fragility of erythrocytes in an aquatic anura, Xenopus laevis, Comparative Biochemistry and Physiology Part A: Physiology 73, no.33 (Jan 1982): 431–436.https://doi.org/10.1016/0300-9629(82)90180-3Martin E. Feder Environmental variability and thermal acclimation of metabolism in tropical anurans, Journal of Thermal Biology 7, no.11 (Jan 1982): 23–28.https://doi.org/10.1016/0306-4565(82)90015-8J. De Costa, M. Alonso-Bedate, A. Fraile Temperature acclimation in amphibians: Changes in lactate dehydrogenase activities and isoenzyme patterns in several tissues from adult Discoglossus pictus pictus (Otth.), Comparative Biochemistry and Physiology Part B: Comparative Biochemistry 70, no.22 (Jan 1981): 331–339.https://doi.org/10.1016/0305-0491(81)90053-5Elizabeth Sherman Cardiovascular responses of the toad Bufo marinus to thermal stress and water deprivation, Comparative Biochemistry and Physiology Part A: Physiology 66, no.44 (Jan 1980): 643–650.https://doi.org/10.1016/0300-9629(80)90012-2K.Y.H. Lagerspitz Interactions of season and temperature acclimation in the control of metabolism in Amphibia, Journal of Thermal Biology 2, no.44 (Dec 1977): 223–231.https://doi.org/10.1016/0306-4565(77)90035-3Richard H. Shertzer, Robert G. Hart, Frank M. Pavlick Thermal acclimation in selected tissues of the leopard frog Rana pipiens, Comparative Biochemistry and Physiology Part A: Physiology 51, no.22 (Jun 1975): 327–334.https://doi.org/10.1016/0300-9629(75)90377-1Mikko N.E. Harri Effect of season, temperature acclimation and starvation upon plasma FFA and glycerol levels in the frog, Rana temporaria, and in the toad, Bufo bufo, Comparative Biochemistry and Physiology Part B: Comparative Biochemistry 50, no.44 (Apr 1975): 531–534.https://doi.org/10.1016/0305-0491(75)90083-8M.N.E. Harri, A. Talo Effect of season and temperature acclimation on the heart rate-temperature relationship in the frog, Rana temporaria, Comparative Biochemistry and Physiology Part A: Physiology 50, no.33 (Mar 1975): 469–472.https://doi.org/10.1016/0300-9629(75)90302-3Mikko N.E. Harri The relation between thyroid activity and responsiveness to adrenaline during cold acclimation in the frog, Rana temporaria, Comparative and General Pharmacology 5, no.3-43-4 (Jan 1974): 305–309.https://doi.org/10.1016/S0010-4035(74)80017-0