Showing papers on "Toad published in 1991"

Water and urea permeability properties of Xenopus oocytes: expression of mRNA from toad urinary bladder

Abstract: The Xenopus oocyte was evaluated as an mRNA expression system for water and urea transporters. Osmotic water permeability (Pf) was measured from the time course of oocyte volume in response to osmo...

Vasopressin depolymerizes F-actin in toad bladder epithelial cells.

Abstract: Vasopressin (AVP) induces the rapid fusion of water channel-containing vesicles with the luminal membrane of its target cell. We have carried out a quantitative study of the F-actin content of toad bladder epithelial cells, using the rhodamine phalloidin binding assay. As early as 1 min after AVP stimulation, there is a significant 15% reduction of cellular F-actin, which remains reduced by 20-30% for the duration of action of AVP. Comparable reductions were seen following 8-bromoadenosine 3',5'-cyclic monophosphate, 1-desamino-8-D-arginine vasopressin, and forskolin. F-actin content rose to and then exceeded that of control bladders after AVP washout. Inhibition of prostaglandin synthesis enhanced both water flow and the decrease of F-actin. In the living cell, stabilization of F-actin with NBD-phallacidin selectively inhibited water flow. In view of the rapidity of the response, we conclude that AVP shifts the equilibrium between F-actin and G-actin monomers, and this depolymerization may be required for vesicle fusion.

Polymorphism in purified guanylate cyclase from vertebrate rod photoreceptors

Abstract: Guanylate cyclase from rod photoreceptors of amphibian (toad, Bufo marinus, and frog, Rana catesbeiana) and bovine retinas was solubilized and purified by a single chromatography step on a GTP-agarose column. Silver staining of purified amphibian enzymes in SDS/polyacrylamide gels disclosed a doublet band (110 and 115 kDa), while the bovine enzyme appeared as a singlet band (110 kDa). The identification of these guanylate cyclases was confirmed using three chromatography systems with the purified enzymes. Specific binding to Con A-Sepharose suggested that rod guanylate cyclase is a glycoprotein. Two-dimensional gel electrophoresis of purified toad, frog, and bovine enzymes resolved two, three, and five variants, respectively, that differed in isoelectric point. Two variants of toad guanylate cyclase showed differences in various characterizations. These data suggest multiple mechanisms for regulation of guanylate cyclase activity in vertebrate rod photoreceptors.

High density cAMP-gated channels at the ciliary membrane in the olfactory receptor cell.

Abstract: Spatial distribution of the cAMP-gated channel was investigated in amphibian olfactory receptor cells. Low doses of cAMP applied to the cytoplasmic side of a membrane patch excised from cilia produced single channel activity of unitary conductance 28pS. Variance analysis showed that the ciliary membrane contained 920 cAMP gated-channels/microns2 in the newt and 2400 channels/microns2 in the toad. In contrast, the membrane of the dendrite and cell body contained only 2 cAMP-gated channels/microns2 (newt) and 6 channels/microns2 (toad). Thus, there is a high density of cAMP-gated channels in the cilia where olfactory transduction is thought to take place.

Angiotensin II Stimulates Cutaneous Drinking in the Toad Bufo punctatus

Abstract: The renin-angiotensin system regulates drinking in many vertebrates but has not been thought to serve this function in amphibians because injections of angiotensin II (AII) have failed to stimulate either oral or cutaneous drinking. We tested the effects of intraperitoneal injections of AII on the duration of water absorption behavior and water weight gain of the red-spotted toad, Bufo punctatus. We found that doses of 1, 5, and 200 μg AII/100 g body weight significantly increased both the duration of water absorption behavior and water weight gain. The effect was eliminated by prior injection of saralasin (a specific competitive inhibitor of AII). This suggests that the mechanism that regulates hydration in amphibians is homologous to thirst mechanisms in other vertebrates and may represent an important step in the evolution of thirst in terrestrial vertebrates.

11-Dehydrocorticosterone, a glucocorticoid metabolite, inhibits aldosterone action in toad bladder.

Abstract: The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) metabolizes glucocorticoid hormones and diminishes their ability to induce sodium transport. In these studies, we determined the location of this enzyme in toad bladder and assessed the biological role for its 11-dehydro end product. Employing a polyclonal antibody directed toward 11 beta-OHSD and immunofluorescence techniques, we located the enzyme in the epithelial cell layer of the toad bladder. Although corticosterone (10(-7) M) can partially suppress aldosterone (10(-7) M)-stimulated short-circuit current (SCC), a clear excess of corticosterone (10(-6) M) did not inhibit the aldosterone-induced induced (10(-8) M) rise in SCC (n = 6). The 11-dehydro product of corticosterone, 11-dehydrocorticosterone (compound A) added to the serosal bath suppressed aldosterone (10(-8) M) peak SCC (360 min) in a dose-dependent fashion reaching 46 +/- 5% of control values at 10(-5) M (n = 6; P less than 0.001). Compound A (10(-5) M) in the mucosal bath also...

Effect of neuropeptide-Y and galanin on autonomic control of heart rate in the toad, Bufo marinus.

Abstract: The effects of neuropeptide-Y (NPY) and galanin (GAL) on the autonomic control of heart rate were investigated in the anaesthetised toad, Bufo marinus. Both vagosympathetic trunks were sectioned to prevent reflex changes in heart rate, and the cardiac responses to electrical stimulation of either the vagal or sympathetic fibres to the heart assessed. Intravenous, bolus doses of 10 or 20 μg (2 or 4 nmol) NPY and 5 or 10 μg (1.5 or 3 nmol) GAL caused pronounced pressor responses but small direct changes in heart rate. Pulse intervals measured after peptide administration were within 5% of control values. All doses of both peptides caused inhibition of action of the cardiac vagus nerves, the maximum inhibition observed in response to 20 μg NPY: mean 49.5 ± 14% (SEM). No significant changes in cardiac sympathetic nerve action were observed. It is concluded that NPY and GAL have similar, important cardiovascular actions in the toad. Similarities between the responses of toads and mammals to NPY suggest a phylogenetic conservation of function for this peptide.

Blockage of na+ currents through poorly selective cation channels in the apical membrane of frog-skin and toad urinary-bladder

Abstract: The blockage of Na+ movements through the poorly selective cation channels in the apical membrane of frog skin (Rana temporaria) and toad urinary bladder (Bufo marinus) was investigated with noise, impedance analysis and microelectrode techniques. Na+ currents through this pathway were studied with NaCl Ringer solutions on both sides. After removal of Ca2+ and other divalent cations from the mucosal compartment, a considerable part of ISC became insensitive to amiloride. In frog skin, the inhibitory effect of amiloride in mucosal Ca2+-free solutions was highly variable. In some experiments a complete lack of inhibition was observed. Similarly, in the absence of amiloride, the inhibitory effect of mucosal Ca2+ varied strongly among frogs. In the absence of mucosal Ca2+, analysis of the fluctuation in ISC revealed a Lorentzian component in the power density spectrum. The corner frequency (fc) of this spontaneous Lorentzian was 12.3 Hz in frog skin and 347 Hz in the toad urinary bladder. In frog skin, nanomolar concentrations of mucosal Ca2+ induced an additional Lorentzian noise component. Its corner frequency shifted upwards with increasing mucosal Ca2+ concentration ([Ca2+]m). The relation between 2πfc and [Ca2+]m was linear at small [Ca2+]m whereas a parabolic increase of fc was observed at the highest [Ca2+]m. In the bladder, nanomolar concentrations of mucosal Ca2+ did not induce an additional noise component but modified the spontaneous Lorentzian noise by increasing fc proportionally with [Ca2+]m. Microelectrode recordings demonstrated that at least part of the Ca2+-blockable current passes through the granulosum cells and confirmed the apical localization of the poorly selective cation channel. The lack of the inhibitory effect of amiloride in Ca2+-free solutions seems to originate from the parallel arrangement of the amiloride- and Ca2+-blockable pathways and from influences of the blockage of apical channels on the basolateral membrane conductances. The latter cross-talk seems to find its origin in the voltage dependence of the basolateral membrane conductance Garty H (1984) J Membr Biol 77:213–222; Nagel W (1985) Pflugers Arch 405 [Suppl 1]:S39–S43}.

Relationship of the aldosterone-induced protein, GP70, to the conductive Na+ channel.

Abstract: Although one of the primary effects of aldosterone is to increase apical membrane Na+ conductance, as yet none of the proteins induced by the hormone in renal epithelia have been shown to be related to the conductive Na+ channel. Because the toad urinary bladder aldosterone-induced glycoprotein, GP70, has recently been localized to the apical surface of this Na+ transporting epithelium, whether GP70 is associated with the Na+ channel was examined. The specificities of a monoclonal antibody used to characterize GP70 (mAb 20) and a polyclonal antibody raised against the purified bovine renal papillary Na+ channel (anti-CH) were compared: GP70 was specifically immunoprecipitated by both mAb 20 and anti-CH. Moreover, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of mAb 20 purified toad urinary bladder membrane preparations was similar to those reported for bovine and A6 cell Na+ channels. Under nonreducing conditions, a single, very large protein was evident; reduction yielded GP70, a 140-kd polypeptide, and a number of minor bands. Interestingly, only GP70 was induced by aldosterone. Thus, GP70 appears to be associated with the toad urinary bladder conductive Na+ channel; whether GP70 is an integral subunit of the channel or whether it functions as a regulatory moiety remains to be determined. Whatever the case, because GP70 is induced by aldosterone, it likely has a central role in Na+ channel modulation.

Characterization of a novel receptor in toad retina with dual specificity for insulin and insulin-like growth factor I.

Abstract: The biochemical properties of insulin receptors from toad retinal membranes were examined in an effort to gain insight into the role this receptor plays in the retina. Competition binding assays revealed that toad retinal membranes contained binding sites that displayed an equal affinity for insulin and insulin-like growth factor I (IGF-I). Affinity labeling of toad retinal membrane proteins with 125I-insulin resulted in the specific labeling of insulin receptor alpha-subunits of approximately 105 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially reduced (alpha beta-heterodimer) receptors affinity-labeled with 125I-insulin indicated the presence of a disulfide-linked beta-subunit of approximately 95 kDa. Endoglycosidase F digestion of the affinity-labeled alpha-subunits increased their mobility by reducing their apparent mass to approximately 83 kDa. This receptor was not detected by immunoblot analysis with a site-specific antipeptide antibody directed against residues 657-670 of the carboxy terminal of the human insulin receptor alpha-subunit, whereas this antibody did label insulin receptor alpha-subunits from pig, cow, rabbit, and chick retinas. In in vitro autophosphorylation assays insulin stimulated the tyrosine phosphorylation of toad retina insulin receptor beta-subunits. These data indicate that toad retinal insulin receptors have a heterotetrameric structure whose alpha-subunits are smaller than other previously reported neuronal insulin receptors. They further suggest that a single receptor may account for both the insulin and IGF-I binding activities associated with toad retinal membranes.

Cyclic AMP increases electrical capacitance of apical membrane of toad urinary bladder.

Abstract: We have measured the effects of oxytocin and three other compounds (chlorophenyl-thio-cyclic AMP, forskolin and theophylline) that increase cytoplasmic cyclic AMP on the impedance of the toad urinary bladder. Membrane capacitance was calculated from transepithelial impedance measured by a computerized sine wave method. All four agents increased tissue capacitance. Since in these tissues this parameter is proportional to apical membrane area our results suggest that cAMP can be a second messenger involved in the action of agents that promote fusion of exocytotic vesicles with the apical membrane.

Neuroendocrine regulation of immunity: the effects of noradrenaline in Xenopus laevis, the South African clawed toad.

Abstract: A functional association between the peripheral nervous and the immune system in Xenopus laevis, the South African clawed toad, is demonstrated. This association involves the neurotransmitter noradrenaline (NA), produced and released by the sympathetic nerves of the spleen.Chemical sympathectomy prior to immunization reduces splenic NA, and decreases thymus-dependent (TD), but increases thymus-independent (TI), antibody responses.Immune challenge with representatives of the three antigen classes affects splenic NA levels differentially. Thus, the modulatory effect of NA on immunity will depend on the immunogen used.Carrier-priming of helper function in TD responses stimulates a transitory NA release in the spleen, while subsequent immunization activates a more prolonged release. The two types of challenge differ in the antigenic dose given. The effects of NA also depend on the time when it is applied. If used early in the in vivo TD response, antibody production is increased, but if given later, suppresso...

Modulation of cytoskeletal organization and cytosolic granule distribution by verapamil in amphibian urinary epithelia

Abstract: The present study examines the role of calcium in modulating epithelial cytomorphology by using verapamil, a calcium antagonist, and considering its effects on cytosolic granule distribution and exocytosis in toad urinary bladder. The effect of verapamil on the detection and distribution of microfilaments in toad urinary bladder using immunogold labeling techniques in toad urinary bladder epithelial cells was also examined. Verapamil, which inhibits antidiuretic hormone (ADH)-mediated water flow, increased the number, size and distribution of dense calcium-containing secretory granules in bladder epithelial cells. This calcium antagonist prevented granule exocytosis, such that, six-times the number of granules were present in verapamil-treated tissues. The normal cytomorphological changes that accompany the actions of ADH were attenuated by verapamil, including ADH-induction of microvilli. ADH increased the number of actin microfilaments as determined using protein A-gold by immunolabeling, whereas, verapamil treatment was unremarkable as compared to control. The results suggest that calcium may play a prominent role in mediating granule exocytosis and membrane fusion events that normally accompany hormone action.

Intracellular pH in the toad Bufo marinus following hypercapnia.

Abstract: Summary We investigated the effects of hypercapnia on intracellular acid-base regulation in brain and liver of the toad Bufo marinus L. After 1 h at 5 % CO2, arterial PCCH increased significantly, from 1.6±0.04 to 5.7±0.23kPa, while brain and liver intracellular pH (pHi) decreased significantly. Reductions in pHi of both tissues were partially compensated by increased levels of bicarbonate. Surprisingly, however, compensation was lower than expected in brain and higher than expected in liver. We suggest that compensation in brain may be limited by secondary effects of bicarbonate loading in this tissue.

Amiloride blockage of Na+ channels in amphibian epithelia does not require external Ca2+.

Abstract: Noise analysis was used to study the influence of external Ca2+ on the blockage of Na+ transport by amiloride. Experiments were done using frog skin (Rana temporaria and Rana catesbeiana), toad urinary bladder (Bufo marinus) and epithelia of A6 cells. In non-depolarized skins and bladders, removal of Ca2+ from the mucosal bath diminished markedly the inhibitory effect of amiloride. Ca2+ depletion also gave rise to the appearance of an additional noise component related to cation movement through the poorly selective cation channel in the apical membrane [Aelvoet I, Erlij D, Van Driessche W (1988) J Physiol (Lond) 398:555–574; Van Driessche W, Desmedt L, Simaels J (1991) Pflugers Arch 418:193–203]. The amplitude of this Ca2+-blockable noise component was elevated by amiloride and markedly exceeded the amiloride-induced Lorentzian noise levels as recorded in the presence of Ca2+. On the other hand, in K+-depolarized skins and bladders as well as in non-depolarized epithelia of A6 cells, the Ca2+-blockable noise was absent or of much smaller amplitude. Depolarization of frog skin and toad urinary bladder apparently inactivated the poorly selective channels, whereas in A6 cells they were not observed. Under these conditions the typical amiloride-induced blocker noise could also be analysed in the absence of Ca2+ and demonstrated that the on and off rates for amiloride binding were not significantly altered by external Ca2+. We conclude that (a) external Ca2+ per se does not affect the inhibitory potency of amiloride, and (b) that the observed differences between frog skin, toad urinary bladder and A6 cells originate from the presence or absence of a poorly selective cation channel rather than from a different amiloride receptor structure.

Effects of dieldrin treatment on physiological and biochemical aspects of the toad embryonic development.

Abstract: Dieldrin is a cylclodiene insecticide highly persistent in nature due to its chemical stability. The exposure of toad embryos to Dieldrin induces hyperactivity in the swimming larvae and inhibition of cholinesterases. However, the inhibition of these enzymes during early development is not life threatening. The present report provides a physiological and biochemical study of the noxious effect of Dieldrin on the toad embryonic development.

Prostaglandin regulation of H+ secretion in amphibian epithelia

Abstract: The role of prostaglandins in regulating H+ excretion in amphibian epithelia was investigated. The abdominal skin of the southern leopard frog Rana pipiens and the urinary bladder of the toad Bufo marinus were used to measure proton excretion across their mucosal surface. Prostaglandin F2 alpha (PGF2 alpha) produced a dose-dependent inhibition of H+ excretion across the frog skin. Frogs pretreated with ibuprofen (30 mg.kg-1.day-1 for 3 days) showed an enhanced proton excretion similar to that observed when frogs are placed in chronic metabolic acidosis. The number of mitochondria-rich cells, the cells responsible for proton excretion, was also increased in frog skins after chronic metabolic acidosis or ibuprofen treatment. Mezerein and the phorbol ester 4 beta-phorbol 12-myristate 13-acetate (4 beta-PMA), activators of protein kinase C (PCK), decreased H+ excretion in frog skin, whereas the inactive phorbol 4 alpha-PMA was without an effect. The inhibition of proton excretion was similar to that observed with PGF2 alpha and suggested that the effects of PGF2 alpha and activation of PKC were mediated through a common pathway. Frogs pretreated with ibuprofen not only had an enhanced proton excretion rate but also had a decrease in cytosolic PKC activity. In another amphibian tissue, the toad urinary bladder, PGE2 inhibited proton excretion at low doses but enhanced H+ excretion at higher doses. Toads maintained under chronic metabolic acidosis had enhanced proton excretion rates and also had a threefold increase in cellular PGE2 concentration, which was consistent with the observation that PGE2 enhanced proton excretion at high doses.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of aldosterone on sodium transport and membrane conductances in toad skin (Bufo viridis)

Abstract: The effect of aldosterone (1 μmol/l for 4–6 h) on Na+ transport across toad skin (Bufo viridis) was studied in skins preincubated in vitro. Short-circuit current (Isc) was consistently and reproducibly elevated in skins from NaCl(100 and 200 mmol/l)-acclimated toads, where the baseline Isc was greatly reduced. The effect of aldosterone was tested in NaCl and NaNO3 Ringer's and also after oxytocin (50 mU/ml) in the latter conditions. Apical membrane conductance of the principal cells increased consistently after aldosterone in all skins and was linearly correlated with the Isc under all conditions. This confirms that the stimulation of Na+ transport originates from the effect of the aldosterone on apical Na+ channels. Basolateral membrane conductance was also significantly elevated compared with control pieces in those tissues that were preincubated with aldosterone for 4–6 h. The increase, however, did not correlate with the magnitude of the Isc. It is therefore concluded that aldosterone specifically stimulates, in addition to the apical effect, the basolateral membrane conductance. This stimulation appears to be direct and not a secondary response to the elevation of transepithelial transport rate.

Effect of estradiol on spermatogenesis and testicular hydroxysteroid dehydrogenase activities in Bidder's organectomized toad (Bufo melanostictus).

Abstract: Inhibition of spermatogenic activity and increase in Leydig Cell nuclear area (L C N A), testicular delta 5-3 beta-hydroxysteroid dehydrogenase (delta 5-3 beta-HSD) and 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activities were noted after Bidder's organectomy. Administration of estradiol in Bidder's organectomized toad showed more or less similar result as the control animal. It is supposed that estradiol coming from the Bidder's organ might play a role in normal testicular activities.

Responses of toad spinal ganglion neurons in vitro to peripheral repetitive stimulation

Abstract: Responses of toad ganglion neurons in vitro to repetitive stimulation of their peripheral processes (sciatic nerve) were intracellularly recorded. The conduction velocity, threshold and resting membrane potential of the 66 recorded neurons ranged 5.3-20.0 m/s, 0.02-0.10 mA, and -50-(-)80 mV, respectively. As the stimulation frequency increased, jitter or delay in onset latency, reduction in amplitude, prolongation in duration, and decrease in afterhyperpolarization occurred in evoked responses of the spinal ganglion neurons. The responses decomposed into three subwaves of S, NM, and M and then missed. The ability of these three components to follow stimulation frequency was in the order of S less than NM less than M; the frequency range during which the spike potential or wave S misses was from 16.7 Hz to 250 Hz. These results indicate that there are three distinct zones in excitability in type A toad spinal ganglion neurons, and some of them may exhibit synaptic or junctional activity.