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Toad (Bufo paracnemius) mast cell degranulation by compound 48/80 and by chlorpromazine: a non-lytic process.

Toad mast cells are degranulated by compound 48/80 and by chlorpromazine. The former was effective at rather high (100 micrograms/ml) and the latter at low (0.1 mM) concentrations. Toad mast cell degranulation induced by these drugs was characterized by cytoplasmic granule dissolution without cell lysis. The metabolic inhibitor, dinitrophenol, blocked the degranulation induced by both agents. Glucose reversed the inhibition of compound 48/80-induced degranulation. The results indicate that toad mast cell degranulation by compound 48/80 and by chlorpromazine is a non-lytic process.

2,4-Dinitrophenol↗

An immunohistochemical study of seasonal changes in luteinizing hormone-releasing hormone and vasotocin in the forebrain and the neurohypophysis of the toad, Bufo japonicus.

Seasonal changes in luteinizing hormone-releasing hormone (LH-RH) and arginine vasotocin (AVT) were examined immunohistochemically in the toad forebrains and neurohypophyses. Strongly immunoreactive (ir-) LH-RH perikarya, from which dense ir-LH-RH fibers project to the median eminence, were localized in the medial septal nucleus and the nucleus of the diagonal band of Broca in the animals captured in the spring and the autumn. While, in the animals collected in the summer, ir-LH-RH perikarya and fibers were sparse, and immunoreactivity in the median eminences was weak. Artificially induced hibernation decreased the density of ir-LH-RH in the median eminence, in contrast with strong immunoreactivity in the control animals kept at room temperature. The amounts of ir-LH-RH in the median eminences of hibernating toads which were captured shortly before the breeding period varied conspicuously among individuals. The median eminences in migrating toads showed relatively weak LH-RH immunoreactivity. After the breeding, the immunoreactivity returned to the strong level that was observed in the spring and the autumn. These seasonal changes in ir-LH-RH seem to correspond to seasonal reproductive activity in this species. However, significant seasonal variations were not found in ir-AVT.

Acclimatization↗

Purification and characterization of toad prolactin.

Prolactin (PRL) was purified from anterior pituitary glands of adult toad (Bufo japonicus formosus) by extraction of acetone-dried pituitary powder with acid acetone and chromatography on DEAE-cellulose and Sephadex G-100. Purification was monitored by radioimmunoassay developed for bullfrog PRL. Toad PRL thus obtained is as potent as bovine PRL in promoting collagen synthesis in the bullfrog tadpole tail fin. HPLC of the toad PRL on a reverse-phase column shows that the preparation behaves as a homogeneous protein. The hormone has a molecular weight of 23,000 as determined by SDS gel electrophoresis. The isoelectric point is 5.5 as determined by analytical electrofocusing. The amino acid composition closely resembles that of bullfrog PRL.

Animals↗

Corticosteroid receptors in liver cytosol of the clawed toad, Xenopus laevis: influence of thyroid and ovarian hormones.

The glucocorticoid receptor capacity Ro and the dissociation constant Kd were determined in the liver of Xenopus laevis by Scatchard analysis. In 5-year-old female toads Ro was about three times higher than that in males (153.9, 54.3 fmol/mg protein) and Kd was similar in both sexes (4.0, 4.1 nM). Some of the animals used had abnormal enlarged thyroid glands, atrophic ovaries, or both defects in connection with different levels of Ro, but not of Kd, compared to those of normal animals. Females with ovarian atrophy showed significantly lower Ro values, in the same range as in normal males, and a high liver weight. In male and female toads with enlarged thyroid glands and in animals with both defects a significantly higher Ro occurred compared to that of the corresponding group without this abnormality. To study the influence of thyroid hormones on glucocorticoid receptors, young toads (2-3 years old) received injections of 4-phenyl-2-thiouracil, T3, or T4 on 7 consecutive days. Ro and Kd were determined on the following day. Doses of 50 and 500 ng T3 and of 500 and 5000 ng T4 per gram of body weight and day resulted in an increase of Ro up to 250% of the controls. Injections of T3 were more efficient in males than in females. The effect of thyroxine was about the same in both sexes. These observations suggest that thyroid and ovarian hormones exert an influence on glucocorticoid receptor capacity and may belong to the factors which regulate glucocorticoid receptors.

Animals↗

Annual cycle of pituitary and plasma gonadotropins and plasma sex steroids in a wild population of the toad, Bufo japonicus.

Pituitary and plasma gonadotropins and plasma sex steroids of free-living toads, Bufo japonicus, were measured monthly from March 1981 to February 1982 and examined in relation to gonadal cycles. Toads were captured at Mizuno, Saitama Prefecture. Individual blood samples were collected by cardiac puncture within 3 min of capture in the field. In males, testicular weight was maximal in August. Plasma follicle-stimulating hormone (FSH) levels changed in association with testicular weight. Plasma androgen levels showed a small peak in November and a large peak in March just prior to breeding. Plasma luteinizing hormone (LH) levels changed in parallel with plasma androgen levels. In females, neither plasma FSH nor LH alone were correlated significantly with ovarian weight. However, ovarian and oviductal weights both correlated significantly with plasma steroid levels. Plasma estradiol levels showed a sharp peak in March, followed by a rapid decrease to the minimum in April. A gradual increase of estradiol occurred from July to November in parallel with an increase in ovarian weight. Changes in plasma progesterone and androgen levels in females resembled those for estradiol. However, the changes in progesterone were not so marked as in estradiol. Plasma androgen levels in females were especially high between January and March. In both sexes, the pituitary gonadotropin contents changed in parallel with plasma levels of both FSH and LH. The pituitary almost always contained more LH than FSH, while the reverse was true in the plasma in both sexes. In addition, plasma FSH levels increased markedly in early summer when plasma LH remained unchanged (males) or increased only slightly (females). These results indicate that the toad may serve as excellent material for the study of differential control of FSH and LH secretion.

Androgens↗

Changes in electric activity (EEG) of the telencephalon of conscious toads (Bufo paracnemis) caused by cholinergic stimulation of the mesencephalic tegmentum.

Studies were carried out to investigate the effect of microinjection of cholinergic agonist carbamylcholine (carbachol) into the mesencephalic tegmentum of conscious toads (Bufo paracnemis), subjected or not to section of the olfactory nerves, on the electrical activity (EEG) of the telencephalon. The response depended on the type of preexisting EEG activity. Marked desynchronization was observed both in animals with intact nerves and in animals with sectioned nerves, whose previous basal activity was already desynchronized (high frequency and low amplitude). In toads with synchronized basal activity and with intact nerves, an increase, a fall or even abolition of preexisting spindle-like activity occurred, whereas in animals with sectioned nerves the effect was reduction or abolition of spindle-like activity. The data show that stimulation of the mesencephalic tegmentum can alter the electrical activity of the telencephalon and that this effect is not only secondary to a change in nasal air flow alterations since it occurs in toads with sectioned olfactory nerves.

Animals↗

Acid-base status in the toad Bufo viridis in vivo.

The acid-base status in the toad Bufo viridis was assessed on blood samples taken through a chronical cannula from unrestrained animals. Blood gases and the pH were very constant in animals which were kept either under control conditions (free access to tap water) or immersed in shallow water. pH was 7.646 +/- 0.032 (mean +/- SEM) in 14 control toads at 26 degrees C. PCO2 was 13.1 +/- 1.2 mm Hg and [HCO3-] was 17.0 +/- 0.7 mmol/l at the same temperature. pH and PCO2 were independent of the hematocrit and the haemoglobin in the blood. Diamox induced a characteristic metabolic acidosis which is apprently the result of the inhibition of carbonic anhydrase both in the kidney and in the skin. The results are discussed in relation to the role of the kidney and the skin in regulating the acid-base balance in the toad.

Acid-Base Equilibrium↗

Hypoxemic threshold for lung ventilation in the toad.

The relationship between the activity of the buccal force pump, expressed as the time integral of positive buccal pressure, and PaO2 was investigated in conscious toads, Bufo marinus, unidirectionally ventilated at a high flow rate (240-260 ml/min). The high ventilatory flow rate meant that PaO2 was largely independent of the animal's ventilatory activity so that the relationship between pulmonary ventilation and PaO2 was effectively open-loop. The hypoxemic threshold (PaO2) for lung ventilation was 54.2 mm Hg in hypocapnia (PaCO2 = 4.7 +/- 0.3 mm Hg), 82.6 mm Hg in normocapnia (PaCO2 = 11.6 +/- 0.2 mm Hg), and 137.9 mm Hg in hypercapnia (PaCO2 = 20.1 +/- 0.1 mm Hg). Unidirectional ventilation with 20% O2 in N2, a condition in which the toads were normoxic but hypocapnic, stopped pulmonary ventilation cycles. Taken with existing evidence that hyperoxia stops pulmonary ventilation even under conditions in which PaCO2 is elevated this suggests that hypoxic and hypercapnic stimuli summate to drive lung ventilation in the toad. Bilateral denervation of the carotid labyrinths decreased pulmonary ventilation in absolute terms, but did not reduce the proportionate increase in pulmonary ventilation in response to normocapnic hypoxia, suggesting that chemoreceptors within the carotid labyrinth may contribute to, but are not solely responsible for, the hypoxemic ventilatory drive.

Animals↗

Toad urinary bladder as a model for studying transepithelial sodium transport.

Sodium ion transport across tight epithelia has been investigated particularly extensively by studying two model systems: the urinary bladder of the toad and the frog skin. The greatest advantage presented by these models is the capability of monitoring net transepithelial Na+ flux simply, precisely, and instantaneously by measurement of the short circuit current (ISC). Many of the caveats involved in the measurement are discussed in detail. In order to fully characterize the forces driving Na+ movement across the series apical and basolateral membranes, it is necessary to measure intracellular potential and ionic composition. Such measurements are far more easily conducted with frog skin than with toad bladder, using the major biophysical techniques currently available. Regulation of transepithelial Na+ movement across tight epithelia is largely conducted at the apical membranes. This regulation can be clarified by study of the isolated Na+ channels in membrane vesicles. Such vesicles are far more easily prepared from toad urinary bladder than from frog skin. The strengths and potential misappropriations of this technique are considered in detail.

Animals↗

Abnormal feeding behavior of western toads (Bufo boreas) kept in a hyperosmotic environment. I. A quantitative behavioral analysis as related to cerebral amino acid concentrations.

Discriminant analysis of eleven behavioral variables associated with feeding permitted the assignment of hyperosmotically-acclimating (HOA) toads (Bufo boreas) to six different behavioral states. These behavioral states could be correlated with specific alterations in the level of select amino acids in three regions of the toad's central nervous system. By considering only those amino acids that showed equivalent levels in corresponding brain regions of normally-behaving, freshwater-acclimating (FWA) and HOA toads, it was possible to focus upon just nine amino acids as possible modulators of feeding behavior. Four of these amino acids were markedly elevated in two or more abnormal behavioral states: gamma-aminobutyric acid (GABA) in all three brain areas; glutamate in the cerebral hemispheres; aspartate in the cerebral hemispheres and olfactory bulbs; and phenylalanine in the olfactory bulbs and optic lobes. Other possible behavior modulators identified were: lysine and threonine in the cerebral hemispheres; carnosine in the olfactory bulbs; and valine and alanine in the optic lobes.

Amino Acids↗

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

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 micrograms (2 or 4 nmol) NPY and 5 or 10 micrograms (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 micrograms 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.

Animals↗

Experimental reorganization in the alar plate of the clawed toad, Xenopus laevis. I. Quantitative and qualitative effects of embryonic otocyst extirpation.

The area octavolateralis and the lateral line projections were examined in larval and postmetamorphic clawed toads, which had one otic vesicle removed at embryonic stage 38 (ref. 20). Premetamorphic tadpoles show a smaller octavolateral area on the operated side as compared to the contralateral control side. This area is in postmetamorphic toadlets significantly reduced as compared to the control side. The largest cells of the magnocellular vestibular nucleus show no significant difference in size to the contralateral side. A dorsolateral auditory nucleus develops prior to metamorphosis and shows in tadpoles no differences in cell size. Cell of this nucleus are in small toads only about 60% the size of cells in the contralateral nucleus. Countings in toadlets indicate a reduction of 40% in cell number on the operated side. Both tadpoles and small toads show virtually no differences in the lateral line projection compared to controls. Only rare collaterals of lateral line fibres can be traced into the neighbouring vestibular and auditory nuclei. The data show no preferential innervation of inner ear afferent deprived auditory and vestibular nuclei by lateral line afferents. In contrast, some collaterals of somatosensory fibres reach into the area deprived of octaval afferents.

Animals↗

Brain creatine kinase activity during ontogeny of the cichlid fish oreochromis mossambicus and the clawed toad Xenopus laevis, influence of gravity?

The development of creatine kinase (CK) activity was studied in the brain of cichlid fish and clawed toads. The activity of CK in the whole brain of the fish decreases immediately after hatching (stage 6) from values of about 135 nmol substrate cleaved/mg protein/min to a value of about 105 at stage 8 (5 days post hatch at 20 degrees C). With the exception of a significant peak (125 nmol) between stages 9 and 10 (7 and 9 days respectively, post hatch at 20 degrees C) and a small intermediate peak at stages 12 and 13 (about 10 days post hatch at 20 degrees C) a constant level of about 100 nmol cleaved substrate is maintained until maturity. In contrast, CK activity was determined to be 3-fold higher in the whole brain of the clawed toad. With the exception of two significant peaks at stages 47 and 49 (5 and 12 days respectively, post fertilization at 23 degrees C) a value of about 360 nmol was found during larval development and metamorphosis, as well as in the adult brain. In investigating the possible influence of gravity on CK activity during early ontogeny of the brain both animal species were exposed to hyper-gravity (3 +/- 1 g) for 7 days. A significant decrease of total CK activity of 20% was found in the fish brain and of about 5% in the toad.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

Angiotensin converting enzyme in the toad Bufo arenarum.

Angiotensin converting enzyme activity (ACEA) was determined in serum, kidney, whole skin and isolated epithelia homogenates of the South American toad Bufo arenarum. ACEA was present in the tissues and serum of the toad. The activity was higher in the kidney, as compared to that of the whole skin or isolated epithelium. Captopril, teprotide and EDTA, caused a significant decrease in the ACEA. Possible physiological roles for the presence of ACEA in the toad are discussed.

Animals↗

Plasma cathepsin activity and the role of hypovolemic shock in dehydrational death in the toad, Bufo marinus.

1. The pH- and activator-dependence of toad plasma cathepsin activity assayed by hemoglobin digestion was characteristic of cathepsins B1 and D. 2. Dehydration, even to the point of death, did not produce a significant elevation of plasma cathepsin activity over controls. 3. Toads were remarkably resistant to the effects of splanchnic artery ligation, which also did not produce significantly higher plasma cathepsin levels. 4. Cardiac depression via the production of a myocardial depressant factor by cathepsins does not appear to be an important factor in dehydrational death in toads.

Animals↗

Determination of transepithelial (mucosal and serosal) electrical potentials in toad skin. Action of chemical agents.

1. Potential differences across the mucosal or outer, and the serosal or inner, membranes of the toad skin (M and S) were recorded separately. Total potential difference across the skin (T) and the short-circuit current (SCC) were recorded by means of the classical Ussing method. 2. The independent determination of the M and the S is of importance in the elucidation of the mechanism of action of agents which alter ion fluxes across the skin. 3. The percentage values of the M and the S obtained in toad skins during the summer were similar to the percentage values obtained by microelectrode impalement of cells. 4. Angiotensin II (AII) and antidiuretic hormone (ADH) increased T with a notable rise in M and a slight increase in S. These agents act mainly by increasing mucosal membrane permeability to Na+ since M is principally affected. 5. Amiloride and ouabain reversed M, decreased T and increased S above T. The reversal of M might be explained by the flow of a cation to the mucosal aspect or of an anion to the cell interior. 6. These results show that the effects of several agents on the toad skin potential may be analysed independently across the mucosal and serosal membranes and reflect the behaviour of the entire tissue rather than of a single cell.

Amiloride↗

Vasotocin and hydrin 2 (vasotocinyl-Gly) in the African toad Bufo regularis: study under various environmental conditions.

1. The neurohypophysial osmoregulatory hormones of the African toad Bufo regularis, a species adapted to estivate under dry and hot conditions, have been investigated. Vasotocin and hydrin 2 (vasotocinyl-Gly) have been identified by their retention times in high-pressure reverse-phase liquid chromatography and coelution with synthetic peptides, their pharmacological properties (vasotocin) and microsequencing. 2. Vasotocin-associated neurophysin (MSEL-neurophysin type) has been characterized by its N-terminal amino acid sequence. 3. In toads subjected to dehydration by evaporation (20% weight loss) or to osmotic stress by immersion in 2% NaCl for 3 hr (6% weight loss), the molar ratio hydrin 2/vasotocin (about 2:1) remained similar to the one observed in control animals. 4. In toads exposed to saline solution, there was a large decrease (roughly 30%) in the amounts of both hormones in the neuro-hypophysis. Environmental conditions for distinct secretions of vasotocin and hydrin 2 remain to be found.

Amino Acid Sequence↗

The influence of ambient salinity and temperature on lipid metabolism in toad (Bufo bufo) skin. Is phosphatidylethanolamine an endogenous regulator of ion channels?

Incorporation of (32P) phosphate and (14C) acetate into frog (Rana temporaria) skin phospholipids in vitro was positively correlated to skin MR cell density. Transport across toad (Bufo bufo) skin and incorporation into skin phospholipids of the radioactive tracers were independent of transepithelial electrical potential in vitro. While all the incorporations in vitro showed (32P) and (14C) frog and toad skin phospholipid patterns dominated by phosphatidylcholine-independent of adaptational temperature and salinity--corresponding phospholipid patterns dominated by phosphatidylethanolamine (PE) were found in vivo, when toads adapted to Ringer solution were transferred to tap water containing tracer amounts of (32P) phosphate and (14C) acetate. PE could play a role in the formation of a "hydrophilic" environment and thereby, e.g. stabilise the integral membrane proteins that regulate the function of ion channels.

Animals↗