Showing papers on "Toad published in 1987"

Cloning and sequence analysis of cDNAs for neurohypophysial hormones vasotocin and mesotocin for the hypothalamus of toad, Bufo japonicus.

Abstract: The primary structures of the precursors of neurohypophysial hormones vasotocin (VT) and mesotocin (MT) in the hypothalamus of the toad Bufo japonicus were determined by analyzing the nucleotide sequences of the cloned cDNAs encoding them. The MT precursor consists of 125 amino acid residues containing a signal peptide followed directly by MT, which in turn is connected to the MT neurophysin by Gly-Lys-Arg, a processing and carboxyl-terminal amidation signal. In contrast, the VT precursor includes a glycoprotein of 36 amino acids following the VT neurophysin. Except for glycoprotein, the structures of MT and VT precursors are quite similar. RNA transfer blotting analysis showed that both MT and VT mRNAs are present in the brain but not in the liver, ovaries, and testes of the toad. The sequences and the structural organizations of the MT and VT precursors are highly homologous to those of their mammalian counterparts, oxytocin and arginine vasopressin precursors, respectively. This fact suggests that, in the evolutionary pathway of neurohypophysial hormones, VT is the ancestor molecule of vasopressin, while MT is that of oxytocin.

The effects of forced activity on circulating catecholamines and pH and water content of erythrocytes in the toad.

Abstract: In vivo experiments were carried out to determine the effect of forced activity on circulating catecholamine levels, haematocrit, and the pH and water content of erythrocytes in the toad, Bufo marinus. In addition, the effect of the beta-adrenergic agonist isoproterenol on erythrocyte pH and water content was examined in vitro. Forced activity caused a significant decrease in both whole blood and erythrocyte pH, while haematocrit and circulating adrenaline and noradrenaline levels increased. Erythrocyte water content did not change following forced activity. Addition of isoproterenol to toad blood in vitro had no effect on either erythrocyte pH or water content. The apparent absence of beta-adrenergic effects on erythrocyte pH and water content in the toad is in sharp contrast to the response of teleost fish erythrocytes to beta-adrenergic stimulation. The significance of these differences is discussed.

Beta-adrenergic relaxation of smooth muscle: differences between cells and tissues.

Abstract: The present studies were carried out in an attempt to resolve the controversy about the Na+ dependence of beta-adrenergic relaxation in smooth muscle. Previous studies on isolated smooth muscle cells from the toad stomach had suggested that at least some of the actions of beta-adrenergic agents, including a stimulatory effect on 45Ca efflux, were dependent on the presence of a normal transmembrane Na+ gradient. Studies by other investigators using tissues derived from mammalian sources had suggested that the relaxing effect of beta-adrenergic agents was Na+ independent. Uncertainty remained as to whether these discrepancies reflected differences between cells and tissues or differences between species. Thus, in the present studies, we utilized both tissues and cells from the same source, the stomach muscle of the toad Bufo marinus, and assessed the Na+ dependence of beta-adrenergic relaxation. We found that elimination of a normal Na+ gradient abolished beta-adrenergic relaxation of isolated cells. In tissues, however, similar manipulations had no effect on relaxation. The reasons for this discrepancy are unclear but do not appear to be attributable to changes in smooth muscle function following enzymatic dispersion. Rather they may be attributable to unique properties of tissues, such as the presence of pacemaker cells, nerves, and so on. Thus the controversy concerning the mechanism of beta-adrenergic relaxation may reflect inherent differences between tissues and cells.

Extrahypothalamic Projection of Immunoreactive Vasotocin Fibers in the Brain of the Toad, Bufo japonicus

Abstract: Extrahypothalamic projection of vasotocin (A VT) fibers in the brain of the toad (Bufo japonicus) was examined immunohistochemically by the avidin-biotin-peroxidase complex (ABC) method. Immunoreactive A VT perikarya are localized in the nucleus preopticus pars magnocellularis. The A VT neurons send their immunoreactive varicose fibers to many discrete brain regions, such as the limbic cortex, the thalamus, the optic tectum and the lower brain stem, in addition to the neurohypophysis. A dense network of A VT fibers was found in the septal nuclei and the anterior part of the preoptic nucleus. A VT fibers which run postero-dorsad project to the nucleus posterocentralis thalami, the nucleus posterodorsalis tegmenti mesencephali, and the nucleus isthmi. Meanwhile, A VT fibers which run through in the dorsal infundibular region and then the mesencephalic reticular formation are distributed in the meduUa oblongata. These findings suggest that A VT acts as a neuromodulator or a local hormone in the toad brain.

Cytosolic calcium and the action of vasopressin in toad urinary bladder.

Abstract: The effects of experimental procedures believed to increase cytosolic calcium on basal and vasopressin-stimulated osmotic water flow and transepithelial sodium transport were examined in the toad u...

A sensitive in vitro toad skin bioassay for melanotropic peptides.

Abstract: 1. An in vitro bioassay for melanotropic peptide utilizing reflectance measurements of toad skin (Bufo ictericus ictericus) is described as an alternative to the commonly used Rana pipiens bioassay. 2. The toad skin bioassay is as sensitive to melanotropins and melanin concentrating hormone (MCH) as the frog bioassay. 3. On the basis of parallel dose-response curves obtained with the toad skin assay we found that beta-MSH is slightly less active than alpha-MSH, whereas the synthetic analogue [Nle4-D-Phe7]-alpha-MSH is about 10 times more potent and exhibits prolonged biological activity. 4. MCH, a putative neurohormone, can also be bioassayed in the in vitro toad skin bioassay, since it has alpha-MSH-like activity on amphibian melanocytes. 5. Since neither adreno- nor cholinoceptors are present in the toad melanocytes, the assay provides great specificity and sensitivity for the determination of melanotropin activity in tissue or blood.

Effects of temperature on contractile properties of skinned muscle fibers from three toad species

Abstract: Single fast fibers were isolated from the iliofibularis muscles of three species of toad with different thermal minima for active locomotion: 8 degrees C, American toad, Bufo americanus; 15 degrees C, Rocky Mountain toad, Bufo woodhousei woodhousei; 22 degrees C, Cane toad, Bufo marinus. All experiments were carried out during the summer. Fibers were chemically skinned and maximum isometric tension and unloaded contraction velocity were determined at a series of temperatures between 0 and 35 degrees C. At 25-30 degrees C, isometric tension development has a low temperature dependence (R10 = 1.1-1.3) and is in the range of 210-260 kN X m-2 for each of the three toads. However, at 0-10 degrees C, absolute values of tension increase in the series (B. americanus greater than B. woodhousei greater than B. marinus; i.e., with increasing cold tolerance), while thermal sensitivity between 0 and 10 degrees C is inversely related to cold tolerance. For example, at 0 degree C, maximum isometric tension (Po) for the most northerly distributed species is three times higher than for the subtropical to tropical species (P less than 0.001). R10 for Po (0-10 degrees C) is 1.7 for B. marinus, 1.3 for B. w. woodhousei, and 1.0 for B. americanus. In contrast, unloaded shortening speeds were similar at any given temperature for the three species. It is concluded that adaptations in Bufo myosin for activity at low temperatures largely involves changes in force production.

Endosomal compartment of toad bladder epithelium

Abstract: Apical exocytosis and increased permeability are induced by antidiuretic hormone (ADH). After this, endocytosis is also induced by ADH and is associated with the decline in ADH-induced water permeability at the apical surface of the toad urinary bladder (9, 19, 20). During this process, horseradish peroxidase (HRP), a fluid phase marker, is taken up from the mucosal solution into endocytic tubules and multivesicular bodies. We now report that we can introduce from the apical (mucosal) side, a viral transmembrane protein (the G-protein of VSV) and that this protein can be retrieved as an integral membrane protein in endocytic membranes. This was demonstrated by immunoisolation of endosomal vesicles loaded with HRP using a monoclonal antibody against the cytoplasmic domain of the G-protein.

Preparation and characterization of the major isotype of parvalbumin from skeletal muscle of the toad (Bufo bufo japonicus)

Abstract: The major isotype of parvalbumin has been isolated from the skeletal muscle of the toad, Bufo bufo japonicus. Unlike the skeletal muscle of every frog so far examined (Rana esculenta, Rana temporaria, and Rana catesbeiana), which contains two major isotypes of parvalbumins, toad skeletal muscle has been shown to contain only one isotype, but the content of parvalbumin in toad skeletal muscle was similar to the sum of those of the two isotypes in skeletal muscles of frogs. This feature of toad skeletal muscle is advantageous to clarify the physiological role of parvalbumin. The relative molecular mass of toad parvalbumin was estimated to be 12,200 by SDS-polyacrylamide gel electrophoresis. The isoelectric point was determined to be 4.81 by polyacrylamide gel isoelectric focusing. The amino acid composition indicated that toad parvalbumin corresponds to bullfrog (R. catesbeiana) pI 4.97 parvalbumin, showing that toad parvalbumin is genetically an alpha-parvalbumin. It was also revealed by the amino acid composition that toad parvalbumin is distinctly different from any of the parvalbumins from frogs. The ultraviolet spectrum of toad parvalbumin is consistent with its amino acid composition. The ultraviolet difference spectrum of the Ca2+-loaded form vs. the metal-free form indicates that some Phe residues in the toad parvalbumin molecule are affected by a conformational change associated with Ca2+ binding. On electrophoresis in polyacrylamide gel in 14 mM Tris and 90 mM glycine, the metal-free and Mg2+-loaded forms of toad parvalbumin migrated twice as fast as the Ca2+-loaded form.(ABSTRACT TRUNCATED AT 250 WORDS)

Phospholipid transfer activities in toad oocytes and developing embryos. [Bufo arenarum]

Abstract: The role of lipid transfer proteins during plasma membrane biogenesis was explored. Developing amphibia embryos were used because during their growth an active plasma membrane biosynthesis occurs together with negligible mitochondrial and endoplasmic reticulum proliferation. Sonicated vesicles, containing /sup 14/C-labeled phospholipids and /sup 3/H-labeled triolein, as donor particles and cross-linked erythrocyte ghosts as acceptor particles were used to measure phospholipid transfer activities in unfertilized oocytes and in developing embryos of the toad Bufo arenarum. Phosphatidylcholine transfer activity in pH 5.1 supernatant of unfertilized oocytes was 8-fold higher than the activity found in female toad liver supernatant, but dropped steadily after fertilization. After 20 hr of development, at the stage of late blastula, the phosphatidylcholine transfer activity had dropped 4-fold. Unfertilized oocyte supernatant exhibited phosphatidylinositol and phosphatidylethanolamine transfer activity also, but at the late blastula stage the former had dropped 18-fold and the latter was no longer detectable under our assay conditions. Our results show that fertilization does not trigger a phospholipid transport process catalyzed by lipid transfer proteins. Moreover, they imply that 75% of the phosphatidylcholine transfer activity and more than 95% of the phosphatidylinositol and phosphatidylethanolamine transfer activities present in pH 5.1 supernatants of unfertilized oocytes may not bemore » essential for toad embryo development. Our findings do not rule out, however, that a phosphatidylcholine-specific lipid transfer protein could be required for embryo early growth.« less

The primary immune response of the green toad (Bufo viridis) to challenge with Crithidia fasciculata.

Abstract: The primary immune response of the green toad (Bufo viridis) following immunization with Crithidia fasciculata choanomastigotes was studied. Lysins, agglutinins, and antibodies detectable by enzyme-linked immunosorbent assay (ELISA) were first detected in the sera of immunized animals one week after injection. The antibody titers increased to significant levels (P less than 0.01) and maximum values were reached seven weeks post-immunization. The stimulated immunoglobulins were antigen-specific, partially heat-labile, sensitive to the reducing agent dithiothreitol, possessed precipitin activity, effectively fixed complement and exhibited an electrophoretic mobility similar to the gamma-globulins of human serum. On this basis, it is probable that the antibody produced during the primary response in green toads is high molecular weight IgM. Increases in serum lysozyme levels paralleled the rise of antibody titers. Overall, the lysozyme concentration increased two-fold compared to the appropriate controls. This is the first report of the immune response in amphibians to experimental injection with protozoan parasites and the use of the ELISA technique to detect antibodies in amphibian sera.

Tyrosination-detyrosination of the COOH-terminus of α-tubulin in oocytes and embryos of Bufo arenarum

Abstract: 1. 1. Soluble tubulin from Bufo arenarum oocytes and early embryos was shown to be composed mainly of the non-tyrosinable species. The low proportion of tyrosinable tubulin was almost exclusively constituted by the tyrosinated form. 2. 2. Compared with oocytes and embryos, toad brain contained a higher proportion of tyrosinable tubulin constituted mainly by the non-tyrosinated form. 3. 3. Tubulin carboxypeptidase was detected in toad brain but not in oocytes and embryos.

Synthesis and biological activities of a photoaffinity probe for vasotocin receptors.

Abstract: This study reports the synthesis and biological activities of 1-desamino, 7-lysine-(4-azidobenzoyl), 8-arginine vasotocin (d7-N3-AVT). This compound was found to be biologically active in the rat antidiuretic assay (20 U/mg), to behave as an antagonist of vasopressin in the rat pressor assay (pA2 = 6.6), and to yield a half-maximal hydroosmotic response in the isolated toad urinary bladder at a bath concentration of 2.4 X 10(-8) M. When toad bladders were exposed to d7-N3-AVT in the presence of long wavelength UV light, the hydroosmotic response persisted in spite of prolonged and repeated periods of washout. By contrast, the hydroosmotic response in control bladders after stimulation with d7-N3-AVT in the absence of UV irradiation was fully reversed within 15 min of washout. A membrane preparation derived from bladders that had been photolabeled with d7-N3-AVT and washed for 1 h specifically bound 325 fmol [3H]vasopressin/mg protein. Matched bladders exposed to the analog in the absence of UV irradiation and washed for 1 h specifically bound 591 fmol [3H]vasopressin per mg of protein. These studies indicate that d7-N3-AVT binds covalently to hydroosmotic receptors of toad urinary bladder and forms a complex that is functional in triggering an increase in the permeability to water of the epithelium. This analog may prove useful in the isolation and purification of vasotocin receptors in lower vertebrates.

Structural and functional response of the isolated toad skin to mucosal lithium.

Abstract: Structural and functional changes induced by long-term Li-exposure of the outer surface of toad skin was studied. Electron microscopy revealed that total Na by Li replacement in the outer compartment of short-circuited toad skin promotes a conspicuous cellular damage expressed as focal swollen cells with altered intercellular spaces and nuclear morphology. Short-circuit current (SCC) decreases by about 70% over the first 60 min after 115 mmol/l Li-exposure. An amiloride sensitive transepithelial Li transport remains intact over a further 150 min despite the epithelial damage, indicating that the pathways across the apical barrier are functioning. Increase of the paracellular permeability is detected by elevation of Na-efflux. Partial 50% or 10% Na by Li replacements induce minor structural alterations and are not sufficient to trigger appreciable Na-efflux and SCC alterations. Therefore, low Li concentrations are less effective than 115 mmol/l in promoting morphofunctional responses. Although ouabain and Li reduce the SCC, ouabain does not promote structural lesions, showing that Li-inhibition of Na transport by itself is not responsible for the observed morphological alterations. In the light of this study, Li utilization as a tool to investigate transepithelial Na transport requires careful judgement.

Ba2+-inhibitable 86Rb+ fluxes across membranes of vesicles from toad urinary bladder.

Abstract: 86Rb+ fluxes have been measured in suspensions of vesicles prepared from the epithelium of toad urinary bladder. A readily measurable barium-sensitive, ouabain-insensitive component has been identified; the concentration of external Ba2+ required for half-maximal inhibition was 0.6 mM. The effects of externally added cations on 86Rb+ influx and efflux have established that this pathway is conductive, with a selectivity for K+, Rb+ and Cs+ over Na+ and Li+. The Rb+ uptake is inversely dependent on external pH, but not significantly affected by internal Ca2+ or external amiloride, quinine, quinidine or lidocaine. It is likely, albeit not yet certain, that the conductive Rb+ pathway is incorporated in basolateral vesicles oriented right-side-out. It is also not yet clear whether this pathway comprises the principle basolateral K+ channel in vivo, and that its properties have been unchanged during the preparative procedures. Subject to these caveats, the data suggest that the inhibition by quinidine of Na+ transport across toad bladder does not arise primarily from membrane depolarization produced by a direct blockage of the basolateral channels. It now seems more likely that the quinidine-induced elevation of intracellular Ca2+ activity directly blocks apical Na+ entry.

Covalent labeling of hydrosmotic toad bladder receptors with an antagonist of vasotocin

Abstract: A photoreactive analogue of vasotocin, [1-desamino,4-lysine(azidobenzoyl),8-arginine]vasotocin (4-N3-AVT), has been examined in the isolated toad urinary bladder for biological activity and binding to hormonal receptors Although 4-N3-AVT induced only a small increase in bladder permeability to water, it behaved as a potent inhibitor of hydrosmotic action of [8-arginine]vasotocin (AVT) and [8-arginine]vasopressin (AVP) The inhibitory action of 4-N3-AVT was readily reversed on removal of the analogue from the serosal bathing solution On the other hand, when bladders were exposed to 4-N3-AVT in the presence of long wavelength UV light (365 nm), the inhibition by 4-N3-AVT was not reversed on washout of the analogue The dose of vasopressin required for a half-maximal response (ED50 value) was increased from 5 X 10(-9) to 13 X 10(-7) M in bladders photolabeled with 4-N3-AVT and the maximal response capacity of the tissue (intrinsic activity) was reduced to 79% of nonphotolabeled controls A crude membrane preparation derived from bladders photolabeled with 4-N3-AVT contained 72 fmol of specific binding sites for tritium-labeled vasopressin per milligram protein, whereas nonphotolabeled controls had 136 fmol of specific binding sites per milligram protein These observations suggest that 4-N3-AVT forms a covalent bond with hydrosmotic receptors in the presence of UV light This is the first antagonistic photoaffinity analogue observed in the toad bladder and it may serve as a useful tool for analyzing the cellular mechanism of action of antidiuretic hormone