Showing papers on "Toad published in 1994"

Evidence for a Ca(2+)-gated ryanodine-sensitive Ca2+ release channel in visceral smooth muscle.

Abstract: Although a role for the ryanodine receptor (RyR) in Ca2+ signaling in smooth muscle has been inferred, direct information on the biochemical and functional properties of the receptor has been largely lacking. Studies were thus carried out to purify and characterize the RyR in stomach smooth muscle cells from the toad Bufo marinus. Intracellular Ca2+ measurements with the Ca(2+)-sensitive fluorescent indicator fura-2 under voltage clamp indicated the presence of a caffeine- and ryanodine-sensitive internal store for Ca2+ in these cells. The (CHAPS)-solubilized, [3H]ryanodine-labeled RyR of toad smooth muscle was partially purified from microsomal membranes by rate density centrifugation as a 30-S protein complex. SDS/PAGE indicated the comigration of a high molecular weight polypeptide with the peak attributed to 30-S RyR, which had a mobility similar to the cardiac RyR and on immunoblots cross-reacted with a monoclonal antibody to the canine cardiac RyR. Following planar lipid bilayer reconstitution of 30-S stomach muscle RyR fractions, single-channel currents (830 pS with 250 mM K+ as the permeant ion) were observed that were activated by Ca2+ and modified by ryanodine. In vesicle-45Ca2+ efflux measurements, the toad channel was activated to a greater extent at 100-1000 microM than 1-10 microM Ca2+. These results suggest that toad stomach muscle contains a ryanodine-sensitive Ca2+ release channel with properties similar but not identical to those of the mammalian skeletal and cardiac Ca(2+)-release channels.

Structures of novel bufadienolides in the eggs of a toad, Bufo marinus.

Abstract: In this paper, we report chemical structures of five compounds including four novel polyhydroxylated cardiac steroids in the eggs of a toad, Bufo marinus. These cardiac steroids were purified by high-performance liquid chromatography, and their structures were determined to be 11 alpha,19-dihydroxy-telocinobufagin (I), 11 alpha-hydroxytelocinobufagin (II), 11 alpha,19-dihydroxymarinobufagin (III), 11 alpha-hydroxymarinobufagin (IV) and 19-hydroxytelocinobufagin (V) on the basis of spectral data of nuclear magnetic resonance and mass spectroscopy. All the five compounds showed biological activity, as tested by inhibition of Na+,K(+)-ATPase activity and of [3H]ouabain binding to the receptor on Na+,K(+)-ATPase. This is the first finding of bufadienolides as cardiac steroids in animal eggs.

Characterization and localization of epithelial Na+ channels in toad urinary bladder

Abstract: The toad urinary bladder and epithelial cell lines derived from the urinary bladder, including TBM, serve as model systems for the study of transepithelial Na+ transport. We examined biochemical characteristics of epithelial Na+ channels in toad urinary bladder and TBM cells and their cellular localization in the urinary bladder. The radiolabeled amiloride analogue [3H]benzamil bound to a single class of high-affinity binding sites in membrane vesicles from toad urinary bladder with a dissociation constant (Kd) of 10 nM. Photoactive benzamil analogues specifically labeled a 135,000-Da polypeptide in toad urinary bladder and TBM cells. A monoclonal anti-Na+ channel antibody directed against the amiloride-binding component of the channel specifically recognized a 135,000-Da polypeptide in TBM cells. Polyclonal anti-Na+ channel antibodies generated against purified bovine epithelial Na+ channel specifically recognized a 235,000-Da polypeptide in toad urinary bladder and localized Na+ channels to the apical plasma membrane of urinary bladder epithelial cells. The biochemical characteristics and the cellular localization of epithelial Na+ channels in toad urinary bladder are similar to those previously described in mammalian kidney and in the A6 cell line.

Further studies on osmotic resistance of nucleated erythrocytes: observations with pigeon, peafowl, lizard and toad erythrocytes during changes in temperature and pH.

Abstract: Summary The osmotic resistance of pigeon, peafowl, lizard and toad erythrocytes at different temperatures and pH was studied. Erythrocytes from female pigeons showed greater osmotic resistance than those from males, but no sex difference appeared with erythrocytes from peafowls. Pigeon erythrocytes were more resistant and the red blood cell, packed cell volume and haemoglobin values were higher than those in peafowls. Although no significant differences appeared in their haemato-logical values, erythrocytes from the lizard were more resistant than erythrocytes from the toad. At higher temperature, the osmotic resistance of pigeon, lizard and toad erythrocytes increased, while that of peafowl erythrocytes decreased. The resistance of toad erythrocytes decreased in acidic and alkaline solutions, but that of peafowl erythrocytes increased in both solutions. However, with pigeon and lizard erythrocytes, the resistance was unaltered in alkaline solution and decreased in acidic solution.

Effects of corticoid agonists and antagonists on apical Na+ permeability of toad urinary bladder

Abstract: Effects of RU-28362 (glucocorticoid agonist), RU-38486 (glucocorticoid antagonist), and RU-26752 (mineralocorticoid antagonist) on the apical Na+ permeability of toad bladder were measured and corr...

Effects of heparin on excitation-contraction coupling in skeletal muscle toad and rat.

Abstract: 1. Intracellularly applied heparin was found to cause a novel, use-dependent block of excitation-contraction (E-C) coupling in skinned skeletal muscle fibres of the toad. After one to four depolarizations in the presence of 100 micrograms ml-1 heparin, no further depolarization-induced responses could be elicited, even though addition of caffeine or lowering [Mg2+] could still induce massive Ca2+ release. This effect could not be reversed by extensive wash-out of the heparin (> 15 min). 2. Heparin (100 micrograms ml-1) did not abolish subsequent depolarization-induced responses if applied while the voltage sensors were in either their resting or inactivated states, that is (a) while a fibre remained fully polarized, (b) when a fibre was already chronically depolarized or (c) after a fibre had been depolarized in the presence of D600 (gallopamil) and then repolarized. 3. When a toad fibre was depolarized in heparin, with the associated Ca2+ release blocked by the presence of 10 mM intracellular Mg2+, subsequent E-C coupling was abolished. Heparin did not interrupt E-C coupling when Ca2+ release was triggered in the absence of any depolarization, by either caffeine or low [Mg2+]. Thus, the opening of the Ca2+ release channels was neither necessary nor sufficient for heparin to abolish E-C coupling. 4. Heparin had direct effects on the contractile apparatus in toad fibres, increasing the Ca2+ sensitivity and decreasing the maximum Ca(2+)-activated force. These effects could only be partly reversed by extensive wash-out of heparin. 5. At 100 micrograms ml-1, both low molecular weight heparin and pentosanpolysulphate, another highly sulphated polysaccharide, were less effective than heparin in blocking the depolarization-induced response and in changing the properties of the contractile apparatus, and these effects could be substantially reversed by wash-out. Two other polyanions, de-N-sulphated heparin (100 micrograms ml-1), which lacked N-sulphate groups, and polyglutamate (500 micrograms ml-1), had no measurable effect on either E-C coupling or the contractile apparatus. 6. In skinned fibres of the extensor digitorum longus muscle of the rat, 100 micrograms ml-1 heparin had little or no effect on E-C coupling and on the Ca2+ sensitivity of the contractile apparatus, but caused a larger reduction of the maximum Ca(2+)-activated force than in skinned fibres of the toad. 7. These results indicate that heparin blocks E-C coupling in toad muscle if, and only if, it is present when the voltage sensors are activated by depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

DFP-sensitive multicatalytic protease complexes (proteasomes) involved in the control of oocyte maturation in the toad, Bufo japonicus.

Abstract: The inhibition of progesterone-induced oocyte maturation by diisopropylfluorophosphate (DFP), a typical serine protease inhibitor, was investigated in oocytes of the Japanese toad Bufo japonicus for the first time. Oocytes to which DFP was externally applied did not undergo germinal vesicle breakdown (GVBD), which is an early signal of oocyte maturation, in response to progesterone. The more inhibitory period was found to be 0–0.5 GVBD50 on a relative time scale [when the time at which 50% of the oocytes had completed GVBD (GVBD50) was set at 1.0], namely, before the beginning of GVBD. DFP-sensitive proteases, which seem to be multifunctional nonlysosomal protease complexes (proteasomes), may already be present in the cytosol of premature oocytes. Peptide hydrolyzing activity, as reflected by proteasome activity, was found to be regulated before and after GVBD. In addition, immunoblotting regarding the native electrophoretic protein profile of the proteasomes throughout the maturational process demonstrated that they undergo alterations in mobility dependent upon the maturational process. These findings raise the possibility that the activities of some endogenous DFP-sensitive proteasomes play distinct, essential roles in oocyte maturation triggered by progesterone in Bufo. © 1994 Wiley-Liss, Inc.

The effect of sex hormones on lipid content and mast cell number in the harderian gland of the female toad, Bufo viridis.

Abstract: The Harderian gland of the toad Bufo viridis is a dimorphic gland owing to the presence of lipid droplets in the female glandular cells present only during summer months. Ovariectomy causes the disappearance of sudanophilia and estrogen-treatment completely prevents this change, while testosterone-injection has little effect. Estradiol-treatment also provokes a proliferation of the interstitial connective tissue concomitantly with the mast cell number increase. Our results suggest that estradiol acts, stimulating both mast cell and connective tissue proliferation, and plays a role in determining the expression of the female type of the toad Harderian gland.

Opioid binding in giant toad and goldfish brain.

Abstract: Opiate receptor expression in phylogenetically different species has played an important role in the study of opioid receptor pharmacology. Total opioid binding measured with the nonselective ligand 3H-diprenorphine reveals a Bmax of 21.7 +/- 1.37 fmol/mg tissue wet wt and a KD of 0.17 +/- 0.03 nM in Bufo marinus (giant toad), as well as a Bmax of 18.17 + 0.41 fmol/mg tissue wet wt and a KD of 0.47 +/- 0.18 nM in Carassius auratus (goldfish). Despite the similar levels of 3H-diprenorphine binding, the composition of binding subtypes in the two species differs. Approximately 30% of total binding corresponds to mu receptors in both species, whereas neither kappa 1 nor delta binding can be detected. However, the remaining 70% of binding differs between the toad and goldfish. In the toad, the non-mu binding corresponds to kappa 2 sites, whereas in the goldfish, the non-mu binding corresponds to kappa 3 sites. The sites can be distinguished biochemically, as well as pharmacologically. After affinity labeling the sites with 3H-NalBzoH, the retention times on an ion-exchange column differ for the peaks of kappa binding in the two species. Although Bufo marinus (giant toad) and Carassius auratus (goldfish) brains express kappa and mu opioid binding, the kappa subtypes in these two species differ.

Plasma potassium may protect sodium pumps of toad hearts from an endogenous inhibitor

Abstract: Resibufogenin (3-hydroxy-14,15-epoxy-20,22-dienolide glycoside) is a potent sodium pump inhibitor present in toad toxin. It is present in the skin of the cane toad (Bufo marinus) at a concentration equivalent to ouabain of approximately 1 mM. Because toads, like other amphibians, have permeable skin, resibufogenin is also found in high concentrations in the blood. In the cane toad the blood concentration is estimated to be 1 microM (D. Lichtstein, S. Kachalsky, and J. Deutsch. Life Sci. 38: 1261-1270, 1986; D. Lichtstein, S. Samuelov, J. Deutsch, H. Xu, R. A. Lutz, S. S. Chernick and S. S. Chernick. Klin. Wochenschr. 65, Suppl. 8: 40-48, 1987), a concentration thousands of times that required to produce toxicity in humans (J. S. Flier, E. Matatos-Flier, J. A. Pallotta, and D. McIsaac. Nature Lond. 279: 341-343, 1979). In examining how the cane toad avoids inhibiting its own sodium pumps, work on the heart showed that 1) cane toads possess a similar number of cardiac sodium pumps as other vertebrates, and 2) normal plasma K+ levels completely prevent ouabain, and presumably resibufogenin, from binding to cardiac sodium pumps of the cane toad. Other species, i.e., rat (Rattus norvegicus) and salamander (Ambystoma mexicanum), did not show K+ protection of their cardiac ouabain binding sites up to normal plasma K+ levels. These species do not possess the high level of endogenous ouabain-like substance found in the toad. K+ demonstrated a capacity to protect the enzymatic activity of the toad heart sodium pumps from the inhibitory effects of ouabain.

Sensory activity in the telencephalon of the clawed toad, Xenopus laevis.

Abstract: In this study data are presented providing evidence that the striatum of the clawed toad, Xenopus laevis, processes mechanosensory, acoustic, and visual information and that multisensory interaction takes place in this telencephalic structure. Multimodal processing as well as the considerable effects of stimulus repetition rate on response amplitudes are in line with the finding that the striatum of Xenopus mediates attentional processes (Traub & Elepfandt, 1990). Further physiological studies are needed to delineate the functional organization of the striatum in amphibians.

The distribution and colocalization of neuropeptides and catecholamines in nerves supplying the gall bladder of the toad, Bufo marinus.

Abstract: The indirect immunofluorescence technique was used to determine the distribution of peptide-containing axons in the gall bladder of the cane toad, Bufo marinus. In addition, the adrenergic innervation of the gall bladder was examined by use of immunoreactivity to the catecholamine-synthesizing enzyme, tyrosine hydroxylase, and glyoxylic acid-induced fluorescence. On the basis of peptide coexistence, two intrinsic populations of neurones and their projecting fibres could be distinguished substance P neurones and vasoactive intestine peptide neurones. Neither of these two types of neurones contained any other colocalized neuropeptides. Four populations of nerve fibres arising from cell bodies outside the gall bladder were identified: nerves containing colocalized galanin, somatostatin and vasoactive intestinal peptide; nerves containing colocalized calcitonin gene-related peptide and substance P; adrenergic nerves containing neuropeptide Y; and nerves containing only adrenaline.

Isolation, purification and partial characterization of a haemolytic protein from bidder's organ of toad bufo melanostictus (schneider)

Abstract: A haemolytic protein toxin (BO-HT) from Bidder's organ of toad, B. melanostictus, purified by DEAE-cellulose column chromatography was electrophoretically homogeneous and was glycoprotein in nature (PAS-positive). The molecular weight was estimated to be 14.4 kDa by SDS-polyacrylamide gel electrophoresis. The sensitivity of the haemolysin of different RBC ghost cell preparation was in the order: buffalo > goat > ox > guinea pig > mice > human > chick > rabbit > rat. The haemolytic activity was increased with the decrease in RBC concentration and was produced over a wide range of temperature. Maximum haemolytic effect was produced at 2 hr of incubation. The toxin showed maximum activity at 3 and minimum at 10 pH. Divalent cations (Ca2+, Zn2+, Cu2+, Mg2+) showed inhibitory effect on BO-HT induced haemolysis, whereas sucrose, EDTA, cholesterol, 2-mercaptoethanol and oxygen did not alter the haemolytic activity. Haemolytic activity was reduced by proteolytic enzymes (trypsin, protease) and was totally antagonized by the toad serum.

Effect of lithium chloride on testicular delta 5-3 beta, 17 beta-hydroxy steroid dehydrogenase, acid phosphatase and gametogenesis in Bufo melanostictus.

Abstract: Injection of lithium chloride at the dose of 200 micrograms/toad/alternate day for 7, 14 and 21 days caused a significant reduction in the activities of testicular delta 5-3 beta, 17 beta-hydroxysteroid dehydrogenase and acid phosphatase enzymes. There was a marked inhibition in spermatogenesis in lithium chloride treated toad for 14 and 21 days of treatment, but 7 days of treatment has no effect.

The gonadotropin-releasing hormone (GnRH) neuron system of the clawed toad Xenopus laevis.

Abstract: The GnRH immunoreactive (GnRH-ir) neuronal system of the Clawed toad Xenopus laevis was studied and compared with the GnRH-ir system of the frog Rana esculenta. Polyclonal antibodies against mammalian (mGnRH) and chicken type-II GnRH (cGnRH-II), and monoclonal antibodies against mGnRH were used in the study. In the Xenopus laevis, most of the immunopositive neuronal cell bodies were located in telencephalic (35-50 per cent) and diencephalic areas (50-65 per cent). About 15-20 per cent of the GnRH perikarya appeared in mesencephalic tegmental regions. Besides the larger GnRH fiber tracts present also in mammals, the toad has rich mGnRH immunopositive axon population in the mesencephalon and in the upper part of the medulla. A similar distribution of the GnRH-ir neuronal elements exists in Rana esculenta, but the number of stained cells and fibers was less. Specificity of the staining of cGnRH-IIir structures located in the lower brainstem could not be proved and therefore the study is only restricted to the findings with mGnRH-antibodies.

Water channel vesicles from toad urinary bladder contain a family of proteins present in other tissues.

Abstract: Antidiuretic hormone (ADH) stimulation causes the fusion and subsequent retrieval of cytoplasmic vesicles containing water channels (WCV) with the apical membrane of toad bladder granular cells. Pr...

Effects of hydrocortisone on acid secretion in the isolated toad gastric mucosa

Abstract: The effects of steroids on gastric acid secretion have been studied in different animal species with contradictory results In the present work we investigated direct effects of hydrocortisone on acid secretion in the isolated toad gastric mucosa (Bufo marinus) The rate of hydrogen ion secretion (QH+) was measured using the pH-stat method of Durbin and Heinz Hydrocortisone significantly stimulated QH+ in a dose dependent manner The effect tended to reach a maximum with the concentration of 10mM and lasted for at least one hour In the mucosa pre-treated with 1mM cimetidine, an specific antagonist of histamine H2 receptors, the addition of 10mM hydrocortisone did not stimulate QH+, but addition of the cholinergic agonist carbachol did produce a significant stimulation in the rate of acid secretion In the gastric mucosa previously stimulated with 5mM hydrocortisone, the addition of a submaximal dose of histamine increased QH+ and this effect was greater than that provoked by histamine in the absence of the steroid The results indicate that hydrocortisone significantly stimulates acid secretion in a dose-dependent manner in the isolated toad gastric mucosa This effect seems to be dependent on the activation of H2 receptors

Expression of desmin in normal and laser (HeNe) irradiated regenerating skeletal muscles in the toad ( Bufo viridis ) and the white rat

Abstract: The expression of desmin was investigated using immunohistochemical methods in normal and low-energy laser (HeNe) irradiated regenerating rat and toad gastrocnemius muscles following partial excision in the former and cold injury in the latter. During the initial stages of regeneration, presumptive myoblasts immunoreacted to desmin antibodies while other mononucleated cells remained unstained. At a later stage the cytoplasm of the myotubes was intensely stained with anti-desmin. At all time intervals there were more mature myogenic structures in the injured zones of the laser irradiated rat or toad muscles than non-irradiated muscles as indicated by the positive immunoreactivity to desmin. It is concluded that desmin immunostaining provides additional information on the role of histological structures during the regeneration process. The process of skeletal muscle regeneration follwing injury is markedly promoted by low-energy direct laser irradiation during the regeneration process. The results also indicate that the rate of differentiation of myoblasts from undifferentiated stem cells is enhanced by the laser irradiation.

Resistive properties of the epithelial membranes of the urinary bladder of the toad, Bufo marinus, determined using the fluorescent dye, RH160.

Abstract: A technique is described for quantitative epifluorescence studies of the apical membrane of the epithelial cells of the urinary bladder of the toad, Bufo marinus, using the lipid-soluble dye, RH160. When the urinary bladder is appropriately mounted, fluorescence signals, in response to a transepithelial voltage pulse, can be recorded from the epithelium immediately after the addition of the dye to the mucosal bath, and for some hours subsequently. The optical signal, recorded as the change in fluorescence in response to a transepithelial voltage pulse, as a fraction of resting fluorescence, was found to be a linear function of the applied voltage over the range ±200 mV, and was approximately 3% for a 100 mV change in transepithelial potential. The signal was enhanced by amiloride (10 μmol · 1−1), reduced by bretylium (5 mmol · 1−1) and abolished in the presence of nystatin (730 U · ml−1). Calculations based on these data permitted estimation of the fractional resistance of the apical membrane, which was found to be 0.85 under control conditions. Apical membrane resistance was 8.6 kΩ · μF, and the basolateral membrane resistance was 1.5 kΩ · μF. These findings support the conclusion that the apical membrane of toad urinary bladder epithelial cells is of high resistance, thus resembling other sodium-transporting epithelia.