Showing papers on "Toad published in 1996"

Effects of reducing agents and oxidants on excitation-contraction coupling in skeletal muscle fibres of rat and toad.

Abstract: 1. The mechanically skinned fibre technique was used to examine the role of oxidation-reduction in the control of Ca2+ release and contraction in rat and toad skeletal muscle fibres under physiological conditions of myoplasmic [Mg2+] and [ATP] and sarcoplasmic reticulum (SR) Ca2+ load. 2. None of the reducing agents, dithiothreitol (DTT, 10 mM), glutathione (GSH, 10 mM) or cysteine (1 and 5 mM), had any detectable effect on the peak force, duration or the total number of depolarization-induced responses that could be elicited in skinned fibres from either toad or rat muscle, except for a slight alteration in one case (GSH on the duration of the response in rat fibres) caused by an effect of the agent of the Ca2+ sensitivity of the contractile apparatus. 3. Application of the reactive disulphide, 2,2'-dithiodipyridine (DTDP, 100 microM), a potent oxidizing agent, never induced any measurable force response or noticeable depletion of SR Ca2+ in any fibre under the conditions used. When all Ca2+ uptake was prevented, DTDP treatment of rat fibres was found to cause a 2- to 3-fold increase in the low rate of Ca2+ "leak' from the SR. DTDP treatment also increased the responsiveness of toad muscle fibres to 1 or 2 mM caffeine. These effects could be largely reversed by treatment with DTT. These results indicate that oxidation of the Ca2+ release channel does not cause substantial channel opening under physiological conditions. 4. Depolarization-induced force responses in both rat and toad fibres were rapidly abolished in the presence of DTDP (10 or 100 microM), in a manner favoured by inactivation of the voltage sensors. The relatively impermeant oxidant, 5,5'-dithionitrobenzoic acid (DTNB, 100 microM), had an effect very similar to DTDP if applied intracellularly, but unlike DTDP, had little or no effect if applied extracellularly (at 5 mM) before skinning. Depolarization-induced responses could be restored by treatment with DTT (10 mM). Intracellular application of the sulfhydryl-alkylating agent, N-ethylmaleimide (NEM, 100 microM), had effects very similar to DTDP and DTNB. 5. These results are not consistent with the proposal that excitation-contraction coupling in skeletal muscle primarily involves the oxidative linkage of the voltage sensors to the Ca2+ release channels, but do show that oxidation of an intracellularly accessible site can interfere with the coupling, in a process made more sensitive by voltage sensor inactivation.

The effect of 2,5-di-(tert-butyl)-1,4-hydroquinone on force responses and the contractile apparatus in mechanically skinned muscle fibres of the rat and toad.

Abstract: In this study, we investigated the effect of the Ca2+ pump inhibitor, 2,5-di-(tert-butyl)-1,4-hydroquinone on the function of the contractile apparatus, Ca2+ uptake, the permeability of the sarcoplasmic reticulum to Ca2+ and excitation-contraction coupling, in mechanically skinned muscle fibres of the rat and toad. 2,5-di-(tert-butyl)-1,4-hydroquinone had no significant effect on the maximum force and Ca2+ sensitivity of the contractile apparatus in rat and toad fibres at concentrations of 20 and 5 μM respectively. In rat fibres, 2,5-di-(tert-butyl)-1,4-hydroquinone was found to inhibit sarcoplasmic reticulum Ca2+ loading in a dose dependent manner, with a half maximal effect at 2 μM. In toad fibres, 5 μM 2,5-di-(tert-butyl)-1,4-hydroquinone completely blocked sarcoplasmic reticulum Ca2+ loading. Exposure to 5 mM BAPTA revealed a small resting sarcoplasmic reticulum Ca2+ leak in unstimulated rat fibres. This Ca2+ leak was not significantly affected by the presence of 20 μM 2,5-di-(tert-butyl)-1,4-hydroquinone, suggesting that 2,5-di-(tert-butyl)-1,4-hydroquinone does not substantially block or activate the sarcoplasmic reticulum Ca2+ release channels. Depolarisation-induced force responses elicited in rat and toad skinned fibres were not significantly affected by 0.5 μM 2,5-di-(tert-butyl)-1,4-hydroquinone. In the rat fibres, 5 and 20 μM 2,5-di-(tert-butyl)-1,4-hydroquinone greatly increased the peak and duration of initial depolarisation-induced force responses, while subsequent responses were reduced. 2,5-di-(tert-butyl)-1,4-hydroquinone did not affect excitation contraction coupling, as depolarisation-induced force responses similar to initial controls could be elicited after 2,5-di-(tert-butyl)-1,4-hydroquinone exposure, provided that the initial Ca2+ release in 2,5-di-(tert-butyl)-1,4-hydroquinone was chelated with 0.5 mM EGTA (to prevent Ca2+-dependent damage) and the sarcoplasmic reticulum was reloaded with Ca2+. In the toad fibres, 5 μM 2,5-di-(tert-butyl)-1,4-hydroquinone had a similar effect on depolarisation-induced force responses to that observed at 20 μM 2,5-di-(tert-butyl)-1,4-hydroquinone in rat fibres. This study shows that 2,5-di-(tert-butyl)-1,4-hydroquinone specifically and reversibly inhibits the sarcoplasmic reticulum Ca2+ pump in skeletal muscle and therefore, 2,5-di-(tert-butyl)-1,4-hydroquinone could be a valuable tool for investigating the role of the sarcoplasmic reticulum in Ca2+ homeostasis in skeletal muscle.

Cloning and Functional Characterization of the Amphibian Mesotocin Receptor, a Member of the Oxytocin/Vasopressin Receptor Superfamily

Abstract: Mesotocin is the oxytocin-like hormone found in most terrestrial vertebrates from lungfishes to marsupials, which includes all non-mammalian tetrapods (amphibians, reptiles, and birds). It has the largest distribution in vertebrates after vasotocin found in all non-mammalian vertebrates and isotocin identified in bony fishes. In this study, we report the cloning and functional characterization of the cDNA for the mesotocin receptor (MTR) from the urinary bladder of the toad Bufo marinus. The cloned cDNA encodes a polypeptide of 389 amino acids that shows the greatest similarity to the teleost fish isotocin receptor and to mammalian oxytocin receptors with mutations in extracellular loops which are involved in ligand binding. When expressed in COSM6 cells, MTR exhibits the following relative order of ligand affinity: mesotocin > vasotocin = oxytocin > vasopressin > hydrin 1, isotocin, hydrin 2. Injection of MTR cRNA into Xenopus laevis oocytes induces membrane chloride currents in response to mesotocin, which indicates the coupling of the mesotocin receptor to the inositol phosphate/calcium pathway. This response is inhibited by an oxytocin antagonist, but not by a vasopressin antagonist specific for V2 vasopressin receptors. MTR mRNA is not only found in toad urinary bladder, but also in kidney, muscle, and brain tissue of the toad as revealed by Northern blot analysis and reverse-transcriptase PCR. The results suggest a variety of functions for mesotocin and its receptor including, in particular, an involvement in the regulation of water and salt transport.

cDNA cloning of a functional water channel from toad urinary bladder epithelium

Abstract: A cDNA was cloned from the epithelium of toad (Bufo marinas) urinary bladder, based on homology to the mammalian aquaporins (AQP). The cDNA [947 base pairs (bp), identified as AQP-t1] encoded a 272...

MARINOIC ACID, A NOVEL BUFADIENOLIDE-RELATED SUBSTANCE IN THE SKIN OF THE GIANT TOAD, Bufo marinus

Abstract: We found a novel substance, 3 beta-hydroxy-11,12-seco-5 beta, 14 beta-bufa-20,22-dienolide-11,14-olide-12-oic acid (1), which we called marinoic acid, in the skin of the toad, Bufo marinus. The structure was established from NMR and MS data. Like bufadienolides, marinoic acid contained an A/B ring structure in the cis configuration and a D/alpha-pyrone ring structure, but the structure of the C ring differed considerably from that of bufadienolides. Marinoic acid exhibited biological activity, as demonstrated by inhibition of Na+, K(+)-ATPase enzymatic activity, and by inhibition of [3H]ouabain binding to the digitalis receptor site on Na+, K(+)-ATPase, although marinoic acid was a less effective inhibitor than typical bufadienolides. Although marinoic acid cannot be classified as a bufadienolide, its chemical structure and its Na+, K(+)-ATPase inhibitory activity suggest that it is bufadienolide-related.

Heterogeneity and lability of endogenous digitalis-like substances in the plasma of the toad, Bufo marinus

Abstract: Three major groups of endogenous digitalis-like substances (EDLS) have been identified in the plasma of the toad, Bufo marinus. One group of compounds, present in fresh plasma, is composed of chromatographically homogeneous polar conjugates, principally bufadienolide 3-sulfates, which exhibit relatively weak Na(+)-K(+)-adenosinetriphosphatase (ATPase) inhibitory activity. A second and larger group of compounds, also found in fresh plasma, includes chromatographically heterogeneous conjugates, which are effective inhibitors of Na(+)-K(+)-ATPase; these compounds possess properties similar to those of bufotoxins. The third group of EDLS consists of free unconjugated bufadienolides, which are also effective Na(+)-K(+)-ATPase inhibitors. These unconjugated bufadienolides are present in relatively low concentrations in fresh toad plasma, but appreciable quantities are enzymatically generated from conjugates (believed to consist principally of bufotoxins) during the in vitro incubation of plasma. We suggest that the extent to which circulating polar EDLS are enzymatically deconjugated in vivo may be important in the regulation of the digitalis-sensitive Na(+)-K(+)-ATPase of toad brain, the only known digitalis-sensitive Na(+)-K(+)-ATPase in the toad.

Cloning of an aquaporin homologue present in water channel containing endosomes of toad urinary bladder

Abstract: Regulation of total body water balance in amphibians by antidiuretic hormone (ADH) contributed to their successful colonization of terrestrial habitats approximately 200-300 million years ago. In the mammalian kidney, ADH modulates epithelial cell apical membrane water permeability (Pf) by fusion and retrieval of cytoplasmic vesicles containing water channel proteins called aquaporins (AQPs). To determine the role of AQPs in ADH-elicited Pf in amphibians, we have identified and characterized a unique AQP from Bufo marinus called AQP toad bladder (AQP-TB). AQP-TB possesses many structural features common to other AQPs, AQP-TB is expressed abundantly in ADH-responsive tissues, including toad urinary bladder and skin as well as lung, skeletal muscle, kidney, and brain. In a manner identical to that reported for the mammalian ADH-elicited water channel AQP2, AQP-TB expression is increased significantly by intervals of dehydration or chronic ADH stimulation. However, expression of AQP-TB protein in Xenopus laevis oocytes does not significantly increase oocyte Pf. The lack of expression of functional AQP-TB water channels in oocytes may result from intracellular sequestration of AQP-TB due to the presence of a YXRF sequence motif present in its carboxyterminal domain.

Gonadotropin-releasing hormone neuroblasts from one olfactory placode can be present in both hemispheres in the clawed toad Xenopus laevis.

Abstract: Ontogenetic differentiation of the GnRH-immunoreactive (GnRHir) neuron system was studied in the clawed toad Xenopus laevis by immunocytochemistry employing polyclonal antibodies ag

Characterization of the atrial natriuretic factor system in lungs of the toad bufo paracnemis

Abstract: Blood pressure in the amphibian pulmonary circulation is relatively high because a single ventricle serves both the systemic and pulmonary circulation, creating a high degree of plasma filtration from pulmonary capillaries. Previous studies have shown that lung atrial natriuretic factor (ANF) may have an important physiological function in preventing edema in mammals. In this study, we report the presence of the complete ANF system in the lungs of the toad Bufo paracnemis. Radioimmunoassay of tissue homogenates revealed that toad lung ANF concentration was approximately twice as high (928.5±83.0 pg mg -1 protein) as that of lung tissue in mammals of a similar size. The amount of ANF was significantly higher in the left than in the right atrium (15.0±1.2 versus 1.9±0.8 ng mg -1 protein; N=4, P<0.001), while the ventricle contained 488.3±41.8 pg mg -1 protein. In extracts of both lungs and atria, highperformance liquid chromatography revealed two forms of the peptide; prohormone and a carboxy-terminal peptide of low molecular mass, which is the biologically active form of peptide. The presence of the prohormone suggests that ANF is synthesized in toad lungs and atria. Characterization of toad lung receptors by a competitive binding assay demonstrated three different subtypes of ANF receptors: the guanylyl cyclase (GC) receptors, GC-A and GC-B, as well as clearance (C) receptors. We conclude that the toad Bufo paracnemis has a well-developed complete ANF system in the lung, suggesting that it has a role in toad lung physiology. Summary

Pharmacological Study of the toad skin extract on experimental animals

Abstract: Toad (Bufo melanostictus) skin a rich source of bioactive compounds, was explored for possible pharmacological effects on experimental animals. The LD50 of toad skin extract (TSE) was found to be 400 mg/kg, (iv) in male albino mice. TSE produced significant hyperthermia and potentiated pentobarbitone induced sleeping time in male albino mice. TSE produced apnoea in cat and rat. It produced a biphasic response, hypotension followed by hypertension in cat and the latter action was blocked by atropine. On isolated heart of guinea pig TSE produced negative chronotropic, positive ionotropic effect and produced reversible blockade of guinea pig auricular contraction. Atropine blocked TSE induced contractile response on guinea pig isolated ileum and rat fundus. Electrical stimulation induced twitch response of chick isolated biventer cervices was inhibited by TSE but it had no effect on the isolated rat phrenic nerve diaphragm preparation. TSE did not possess haemorrhagic activity but produced strong haemolytic activity.

[Serotonin inhibits phorbol ester-induced oocyte maturation in the green toad (Bufo viridis)].

Abstract: The activator of protein kinase C phorbol-12-myristat-13-acetate (PMA) induces maturation of the definitive intact (coated with the follicular envelopes) and defolliculated oocytes of the green toad. This effect is more pronounced in case of defolliculated oocytes. The amount of matured oocytes depends on the PMA concentration in solution. Serotonin (5-HT) inhibits or blocks maturation of the oocytes induced either by progesterone or by PMA. A possible mechanism of this effect is discussed with special reference to its role in regulation of oocyte maturation.

Effects of angiotensin II antagonists on the contractile and hydrosmotic effect of AT II and AT III in the toad (Bufo arenarum)

Abstract: The effects of peptide and non-peptide angiotensin II receptor antagonists on the responses to angiotensin II were examined using aortic rings and skin isolated from the toad. The contractile responses of aortic rings to (Ala-Pro-Gly) angiotensin II were inhibited by angiotensin II analogue Leu8 angiotensin II, with a pA2 value of 7.6. Similarly, the concentration response curve for (Ala-Pro-Gly) angiotensin II was displaced to the right by the specific angiotensin receptor subtype antagonist DuP 753, with a pA2 value of 6.0. In contrast, the angiotensin receptor subtype 2 antagonists PD 123177 and CGP 42112A did not modify the contractile response to (Ala-Pro-Gly) angiotensin II. None of the antagonists was able to alter the contractile response to norepinephrine. Both Leu8 angiotensin II (10(-8) mol.1(-1)) and DuP 753 (10(-6) mol.1(-1)) partially inhibited angiotensin III-induced contractions in toad aorta. Angiotensin III, in turn, exhibited lower activity than [Asn1-Val5] angiotensin II in this preparation, its molar potency ratio being 0.293. Previous work from this laboratory reported that osmotic water permeability in the skin of the toad Bufo arenarum was increased by angiotensin II, the effect being blocked by the peptide antagonist Leu8 angiotensin II. The hydrosmotic response to [Asn1-Val5] angiotensin II(10(-7) mol.1(-1)) was significantly inhibited by DuP 753 (10(-6) and 5 x 10(-6) mol.1(-1)), whereas the response was not inhibited by a tenfold higher concentration of either PD 123177 or CGP 42112A. DuP 753 (10(-6) mol.1(-1) also inhibited the hydrosmotic response to angiotensin III (10(-7) mol.1(-1)). These results suggest that receptors for angiotensin II present isolated toad aorta and skin exhibit pharmacological features similar to those characterized as angiotensin subtype 1 in mammalian tissues.

Atrial natriuretic factor (ANF)-like immunoreactivity and secretory granules in internal gills tadpoles of South American toad Bufo arenarum (Hensel).

Abstract: In this study, an ultrastructural and immunohistochemical analysis was carried out on the presence of Atrial Natriuretic Factor-like substance in the internal gills of premetamorphic larval stages of the South American toad. The results of immunohistochemical study indicate that, at larval 27 and following stages, ANF-like immunoreactive cells are seen in internal gills. At electron-microscopy level "secretory granules" similar to those ANF-immunoreactives granules of myoendocrine cells of the same species are seen only in clear cells of gill rakers. Our results reveal the occurrence of ANF-like substance in Bufo arenarum gills and may suggest that "cardiac hormones" might play a paracrine and/or autocrine role in the water and electrolyte balance at internal gills level.

[Blocking effects of benzyltetrahydropalmatine on delayed rectified K+ currents expressed in Xenopus oocytes and in toad oocytes].

Abstract: The effects of benzyltetrahydropalmatine (BTHP) on delayed rectified K+ currents (Ik) expressed in Xenopus oocytes and Ik of toad (Bufo bufo gargarizans) oocytes were studied. The Ik expressed in Xenopus oocytes was measured after microinjection of mRNA isolated from carp fish (C anratus L.) brains with double -microelectrode voltage clamp technique. The maximum and mean value of Ik expressed in Xenopus oocytes were 600 nA and 360 +/- 104 nA, respectively. BTHP reduced the current amplitude of Ik expressed in Xenopus oocytes in 10-1000 mumol.L-1 dose-dependently, EC50 was 29 mumol.L-1. Also, the reduction of Ik of toad oocytes was 9.1%, 29.1%, 54.7% and 68.6% by BTHP 10, 30, 100 and 1000 mumol.L-1, respectively, EC50 was 33 mumol.L-1. The results showed that BTHP possesses an inhibitory effect on Ik, the main ion mechanism of antiarrhythmic action of BTHP.