The muscle fatty acid binding protein of spadefoot toad (Scaphiopus couchii)

Fatty acid binding protein was purified from skeletal muscle of the spadefoot toad (Scaphiopus couchii), an estivating species. While estivating, this animal relies on the fatty acid oxidation for energy. Hence we were interested in the behaviour of fatty acid binding protein under conditions of elevated urea (up to 200 mM) and potassium chloride such as exist during estivation. Also we examined whether there were interactions between glycolytic intermediates and the binding ability of the protein. The amount of bound fatty acid (a fluorescence assay using cis-parinarate) was not affected (P < 0.05) by glucose, fructose 6-phosphate or phosphoenolpyruvate at physiological concentrations. By contrast, glucose 6-phosphate increased the amount of bound cis-parinarate but the apparent dissociation constant was not different from the control. Fructose 1,6-bisphosphate but not fructose 2,6-phosphate decreased cis-parinarate binding by 40%, commensurate with doubling the apparent dissociation constant (1.15-2.62 microM). Urea, guanidinium and trimethylamine N-oxide at 200 mM increased cis-parinarate binding 60% over controls. Urea (1 M) and KCl (200 mM) did not affect cis-parinarate binding compared to controls. The interaction of this fatty acid transporter with fructose 1,6-bisphosphate is discussed in terms of reciprocal interaction with phosphofructokinase since fatty acid is also an inhibitor of phosphofructokinase.     

Characterization of microsatellite markers for Couch's spadefoot toad (Scaphiopus couchii) and cross‐amplification in other species of the family Scaphiopodidae

I developed 12 di‐ and tetranucleotide microsatellite loci for Couch's spadefoot toad (Scaphiopus couchii). These loci have 3–37 alleles per locus and observed heterozygosities ranging from 0.157 to 0.941 among 85 individuals from four populations. Global and within‐population exact tests do not reveal departure from Hardy–Weinberg expectations and all loci pairs are in linkage equilibrium. These independent markers will be useful for studies of population structure and kinship in this commonly studied amphibian. Additionally, several of these loci may be applicable for studies of other North American toads of the family Scaphiopodidae.     

Reversible phosphorylation control of skeletal muscle pyruvate kinase and phosphofructokinase during estivation in the spadefoot toad,Scaphiopus couchii

Both pyruvate kinase (PK) and phosphofructokinase (PFK) occur in two different forms, separable by isoelectric focusing (IEF), in skeletal muscle of the spadefoot toad Scaphiopus couchii. During estivation (aerobic dormancy) the proportions of the two forms changed compared with controls; in both cases the amount of enzyme in Peak I (pI = 5.3-5.4) decreased whereas activity in Peak II (isoelectric point = 6.2-6.4) increased. In vitro incubation of crude muscle extracts with 32P-ATP under conditions that promoted the activity of cAMP-dependent protein kinase led to strong radiolabeling associated with Peak I, but not Peak II, and reverse phase HPLC confirmed that 32P was associated with the subunits of both PK and PFK found in Peak I. Specific radiolabeling of Peak I PK and PFK by protein kinase A was further confirmed using immunoprecipitation. In total, this information allowed identification of the Peaks I and II enzymes as the phosphorylated and dephosphorylated forms, respectively, and the effect of estivation was to increase the proportion of dephosphorylated PK and PFK in muscle. Analysis of the kinetic properties of partially purified PK and PFK revealed significant kinetic differences between the two forms of each enzyme. For PK, the Peak II (low phosphate) enzyme showed a 1.6-fold higher Km for phosphoenolpyruvate and a 2.4-fold higher Ka for fructose-1,6-bisphosphate than did the Peak I (high phosphate) form. These kinetic properties suggest that Peak II PK is the less active form, and coupled with the shift to predominantly the Peak II form during estivation (87% Peak II vs. 13% Peak I), are consistent with a suppression of PK activity in estivating muscle, as part of the overall metabolic rate depression of the estivating state. A similar shift to predominantly the Peak II, low phosphate, form of PFK (75% Peak II, 25% Peak I) in muscle of estivating animals is also consistent with metabolic suppression since phosphorylation of vertebrate skeletal muscle PFK is typically stimulated during exercise to enhance enzyme binding to myofibrils in active muscle. Peak II PFK also showed reduced sensitivity to inhibition by Mg:ATP (I50 50% higher) compared with the Peak I form suggesting that the enzyme in estivating muscle is less tightly regulated by cellular adenylate status than in awake toads. The data indicate that reversible phosphorylation control over the activity states of enzymes of intermediary metabolism is an important mechanism for regulating transitions between dormant and active states in estivating species.     

Postembryonic ontogeny of the spadefoot toad,Scaphiopus intermontanus (Anura: Pelobatidae): Skeletal morphology

Postembryonic skeletal ontogeny of the pelobatid frog Scaphiopus intermontanus is described based on a developmental series of cleared-and-stained, whole-mount specimens. The focus is on laboratory-reared individuals fed a herbivorous diet as larvae. Although there is variation in the timing of ossification of individual skeletal elements relative to developmental stages based on external morphological criteria, the sequence of skeletal development generally is conservative. Compared with its close relative, S. bombifrons, ossifications that occur during prometamorphosis tend to be slightly delayed in S. intermontanus; however, cranial bones that ossify during late metamorphic climax in S. intermontanus are delayed until postmetamorphosis in S. bombifrons. The differences in timing between the two species are consistent, however, with differences observed between two developmental series of S. intermontanus raised at two different temperatures. Noteworthy features of skeletal development in S. intermontanus include: 1) presence of palatine ossifications that form from independent centers of ossification and soon fuse with the postnarial portion of the vomers to form the compound vomeropalatine bones; 2) compound sphenethmoid that may arise from four or more endochondral centers of ossification and one dorsal, dermal center of ossification; and 3) presence of transverse processes and vestigal prezygapophyses on the first postsacral vertebra. The morphology of the larval orbitohyoideus and interhyoideus muscles is compared. The record of skeletal ontogeny and muscle morphology presented herein for the herbivorous larval morph can serve as a baseline for comparisons with the ontogeny of the carnivorous larval morph of Scaphiopus. J. Morphol. 238:179–244, 1998. © 1998 Wiley-Liss, Inc.     

Antioxidant defenses and lipid peroxidation damage in estivating toads, Scaphiopus couchii

Tissue-specific changes in antioxidant defenses and lipid peroxidation damage were analyzed in spadefoot toads, Scaphiopus couchii, to determine how these responded during estivation, a state of suppressed oxygen consumption. Maximal activities of glutathione-S-transferase, glutathione reductase, glutathione peroxidase, superoxide dismutase and catalase were measured in six organs from 2-month-estivated toads and compared with activities in animals awakened for 10 days after estivation. Activities of many enzymes, particularly the glutathione-linked enzymes, were significantly lower in tissues of estivating toads than in awake toads. This indicates that enzymatic antioxidant defenses are probably modulated in response to the rate of reactive oxygen species generation in tissues, which is proportional to oxygen consumption. Antioxidant enzyme activities were largely insensitive to high urea, which accumulates during estivation, but were inhibited by elevated KCl. Levels of reduced glutathione were also significantly lower in three organs during estivation and all organs, except skeletal muscle, exhibited a higher oxidized/reduced glutathione ratio, indicating a more oxidized state during estivation. Products of lipid peroxidation (conjugated dienes, lipid hydroperoxides) were higher in tissues of estivated than control toads, suggesting accumulated oxidative damage to lipids during dormancy. One enzymatic source of free radical generation, xanthine oxidase, appeared to have little impact because its activity was detectable only in liver and was significantly lower in estivated toads. The data indicate that both enzymatic and metabolite antioxidant defenses in toads are adaptable systems that are modulated in estivating versus awake states. Accepted: 21 October 1997     

The Influence of Parasite Infection on Mating Success in Spadefoot Toads, Scaphiopus couchii

The desert toads, Scaphiopus couchii, have an annual activityseason of less than 8 weeks and experience only one significantparasite infection: the monogenean Pseudodiplorchis americanusis transmitted during host spawning and provides a natural systemfor testing the influence of parasite burden on host matingsuccess. The 10 month hibernation involves total starvationduring which the blood-feeding parasites reduce fat reservesand haematocrit. The toads emerge and spawn on the first nightafter rainfall, before they replenish depleted reserves. Malechorusing is energetically very demanding and mate selection,limited to a 7 hr nocturnal assembly, is determined principallyby female choice. Parasite transmission triggered by host sexualactivity results in 100% prevalence and high intensities ofinfection amongst males. Around 50% of toads lose their burdensentirely but the rest carry chronic infections throughout hosthibernation. Field data show a consistent reduction in intensityeach year strongly suggesting resistance. Parasite infectionis pathogenic and creates extra stress during hibernation; therefore,to the extent that elimination of infection is heritable, toadsentering spawning assemblies with heavy burdens should makepoor mates. However, extensive field studies show no correlationbetween mate success and parasite burden. Although infectioncan prejudice survival, it is only one of several inter-relatedfactors (including feeding success, tolerance of hibernation).The condition of successful and unsuccessful males in spawningassemblies indicates that all exceed a threshold at which parasite-inducedpathology is significant. Males which are debilitated by infection—orother factors—are selected against before mate choicebegins     

Effects of changes in plasma hepatic-portal and systemic osmolality on plasma concentrations of arginine vasopressin in conscious newborn calves

Systemic plasma concentrations of arginine vasopressin (AVP) were studied in three groups of 10-15 day-old conscious newborn calves. Animals in the first group (control group) and in the second group (systemic-hypertonic-injected group) received respectively isotonic and hypertonic (8 mmol NaCl/kg body weight) saline injection into the right jugular vein. Animals in the third group were fitted with chronic mesenteric and hepatic-portal catheters and received a 1 h-hypertonic saline infusion (2 mmol NaCl/kg body weight) into the main mesenteric vein. In animals in the second group there were parallel increases in systemic plasma concentration of Na+ (from 148.0 +/- 2.6 to 177 +/- 8 mmol/l; P less than 0.01), osmolality (from 289 +/- 2 to 319 +/- 4 mOsmol/kg H2O; P less than 0.01) and systemic plasma concentrations of AVP (from 4.2 +/- 0.4 to 11.1 +/- 0.6 pmol/l; P less than 0.01) 10 min after the injection. There were no significant changes in control animals. Hypertonic saline infusion into the main mesenteric vein in the third group induced an increase in concentration of Na+ (from 147.3 +/- 2.0 to 165.0 +/- 5.0 mmol/l; P less than 0.01) and osmolality (from 288 +/- 5 to 315 +/- 10 mOsmol/kg H2O; P less than 0.01) in hepatic-portal vein plasma but did not alter systemic plasma osmolality or concentrations of Na+ and AVP. This study demonstrates that the relationship between plasma concentrations of AVP and systemic osmolality is operative in the newborn calf but does not support the hypothesis that hepatic portal osmo-receptors sensitive to hyperosmolality influence AVP release.     

Effects of angiotensin II and bladder condition on hydration behavior and water uptake in the toad,Bufo woodhousei

• 1.1. Water absorption response (WR) behavior and water weight gain were examined in hydrated toads, Bufo woodhousei, treated with angiotensin II (All) or with a control Ringer's solution. The effects of urinary bladder condition (ad lib. bladder urine or empty bladder) were examined concurrently.
• 2.2. Toads treated with All (100μg/100g body weight), spent more time in WR posture and absorbed more water than Ringer's-injected toads.
• 3.3. Toads with empty bladders maintained WR posture for longer periods of time and gained more weight than toads whose bladders were not emptied.
• 4.4. The effects of All and bladder urine on water absorption by B. woodhousei appear to be separate and additive.

     

盐碱胁迫对青海湖裸鲤耗氧率、血浆渗透浓度和离子浓度的影响

采用呼吸生理和血液生理的方法,探讨了性成熟青海湖裸鲤(Gymnocypris przewalskii)对盐度、碳酸盐碱度的适应性以及青海湖裸鲤在盐碱胁迫下的渗透调节和离子调节规律。将裸鲤从淡水突变到盐水(盐度14.01±0.01)、碱水(碳酸盐碱度33.03±0.01mmol·L-1)和青海湖水(盐度12.12±0.02、碳酸盐碱度29.07±0.09mmol·L-1)中,测定了在不同水质胁迫下裸鲤耗氧率、血浆渗透压和离子浓度的变化。结果表明:与淡水对照组比较,呼吸耗氧率除了盐度组在3h有显著性升高(P<0.05),其他实验组96h内均未出现显著性变化(P>0.05);裸鲤的窒息点为0.14～0.17mg·L-1,各实验组窒息点均没有显著性差异(P>0.05);碱度组血浆的渗透浓度和Na+、Cl-、Ca2+离子浓度在96h内未出现变化(P>0.05),盐度组和青海湖水组在所测时间段有显著的升高(P<0.05),并且随着胁迫时间的延长,在72h达到峰值。各实验组血浆K+浓度变化没有血浆Na+、Cl-明显,K+浓度有增高的趋势,且都在72h达到了峰值。青海湖裸鲤具有较强的盐碱耐受性,能通过渗透调节和离子调控适应高盐碱环境,而消耗能量较少。     

The effects of in vivo and in vitro hyperosmolality on skeletal muscle performance in the amphibians Rana pipiens and Scaphiopus couchii

1. The effect of in vivo and in vitro hyperosmolality on skeletal muscle function was investigated in two species of anuarans Scaphiopus couchii and Rana pipiens. 2. Muscle contractile performance, measured as peak tetanic tension declined to greater degree when tissue dehydration occurred in vitro rather than in vivo, even though tissue water contents were greater in vivo. 3. The muscles from S. couchii, a more dehydration tolerant species than R. pipiens, maintained tension at lower tissue water contents than R. pipiens. 4. Data for the effects of in vivo dehydration on plasma sodium, urea and osmotic concentration, as well as tissue water contents, are also presented for both species.     

Taurine in toad brain and blood under different conditions of osmolality: An immunohistochemical study

The concentrations of taurine in blood and brain regions of the toadBufo boreas have been measured. Most of these values are considerably lower than those found in mammals. Using an antibody prepared against conjugated taurine, the distribution of taurine in three brain regions of the toad has been visualized. The possible osmoregulatory functions of taurine have been investigated by making toads hyper- or hypo-osmotic in vivo. Induction of hypoosmolality is accompanied by a massive taurine tide in blood plasma, but has no immediate effects upon the taurine concentrations in the brain areas studied. However, histochemical visualization indicates a marked redistribution of taurine between cellular components and extracellular space of brain tissues. This may indicate that taurine has an osmoregulatory function in brain tissue under hypo-osmotic conditions. Hyperosmolality results in no elevation of the taurine concentration in blood plasma of toads, but rather in a very gradual decline of total plasma taurine content over a prolonged time period. Histochemical studies reveal little change in frontal cortex after 1 hour but deeper staining of many neurons in optic lobe accompanied by greater staining in the extracellular fluid. By 3 hours there is a depletion of taurine from all compartments of cerebral cortex tissues. No evidence of any prolonged direct osmoregulatory role for taurine is indicated under hyperosmotic conditions. A possible indirect osmoregulatory function of taurine is discussed.Special issue dedicated to Dr. Claude Baxter.     

Variation in the effects of antidiuretic hormone on the isolated skin of the toad, Scaphiopus couchi.

The rate of active sodium transport as measured by short-circuit current across the isolated skin of the toad, Scaphiopus couchi, was elevated following vasopressin (0.2 units/ml) or arginine vasotocin (0.1 units/ml) treatment of skins from active animals at all times of the year tested. Skins from dormant animals showed no such elevation at any time of the year. The rate of active sodium transport was elevated following treatment with dibutyryl cyclic AMP (2.5mM) plus theophylline (10 mM) in all skins tested. The hydraulic conductivity of isolated skins from both active and dormant animals showed no significant change following treatment with vasopressin (0.2 units/ml) or arginine vasotocin (0.1 units/ml except on the first day following emergence from dormancy in the field. A correlation was, therefore, observed between the occurrence of a hydroosmotic response to antidiuretic hormones and the seasonal exposure of S. couchi to standing water. A small but significant elevation of hydraulic conductivity was observed across the skins of dormant toads following treatment with dibutyryl cyclic AMP (2.5 mM) plus theophylline (10 mM) whereas a substantial elevation was observed with the skins of active animals.     

Effects of angiotensin II, arginine-vasopressin, endothelin and catecholamines on plasma atrial natriuretic peptide concentrations in the conscious newborn calf.

The effects of epinephrine (E), norepinephrine (NE), angiotensin II (AII), arginine-vasopressin (AVP) and endothelin on plasma ANP levels were studied according to a latin square design in six 12-21 days-old conscious Jersey calves weighing 30 +/- 4 kg. The animals chronically-instrumented with a carotid catheter for blood pressure recording, received at 11.00 a.m. an i.v. right jugular continuous infusion for 30 min of two different sub-pressor or pressor dose-levels of each substance; E: 0.6 and 5.5 nmol/min per kg body wt; NE: 0.6 and 6 nmol/min per kg body wt; AII: 9.6 and 96 pmol/min per kg body wt; AVP: 0.6 and 69 pmol/min per kg body wt; and endothelin: 1.2 and 12 pmol/min per kg body wt). Control animals received, in the same way, the same volume (2 ml/kg body wt) of NaCl 0.9%. In Jersey calves, basal plasma atrial naturetic peptide (ANP) levels were around 5 pmol/l. Marked increases in this parameter were produced by all substances when given at the highest dose-level. The maximal rise of 600% was observed with AII; however on a molar basis, endothelin appeared more potent than AII and at the same dose-level, E appeared more effective than NE to increase circulating ANP (17.8 +/- 0.3 vs 9.5 +/- 0.1 respectively at time 70 min; P less than 0.01). The time-course of plasma ANP levels was positively correlated (P less than 0.01) by linear regression with the increase in blood pressure when pressor agents were given at the highest dose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intra- and extracellular dehydration has no effect on plasma levels of angiotensin II in an amphibian

Previous studies have demonstrated that both dehydration (intra and extracellular) and treatment with angiotensin II (A-II) induce changes in thirst-related behavior in the spadefoot toad, Scaphiopus couchii. One of the steps in determining a causal relationship between a hormone and a behavior is to determine that there is association between an animal's performance of the behavior and changes in endogenous hormonal concentrations. The hypothesis tested that plasma levels of the peptide hormone A-II would change as a result of dehydration known to induce water absorption response (WR) behavior in the spadefoot toad. Plasma samples were taken from toads dehydrated intracellularly by injection of hypertonic solutions of NaCl or sucrose at levels known to induce WR behavior. As an osmotic control, a group of animals was injected with urea, which has been demonstrated to not induce WR behavior. In order to determine the effects of extracellular dehydration on plasma, A-II levels in toads dehydrated by plasma volume depletion via cardiac puncture were compared to sham-punctured controls. None of the treatments in any experiment resulted in significant differences in plasma levels of angiotensin II among groups sampled at the time when WR behavior occurs. These results do not support the hypothesis that dehydration-induced thirst is stimulated by changes in plasma A-II concentrations at the onset of WR behavior. J. Exp. Zool. 286:343-349, 2000.     

Effects of angiotensin II on isolated toad (Bufo arenarum) aortic rings

1. The vascular response to Asn1-Val5 angiotensin II (A II) in aortic rings from Bufo arenarum toad was studied. 2. Tachyphylaxis in response to A II could be abolished after incubation with norepinephrine (NE). 3. Phentolamine treatment partially inhibited the pressor effects to A II. 4. Sar1-Ile8 A II and Sar1-Ala8 A II significantly attenuated the vascular effects of A II and did not affect the NE response. 5. We conclude that the pressor response to A II has a direct contractile effect and a catecholamine dependent component in aortic rings of Bufo arenarum toad.