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PubMed1970

Effects of prolonged saline exposure on water, sodium and urea transport and on electron-microscopical characteristics of the isolated urinary bladder of the toad Bufo bufo.

1. A comparison was made of various transport properties and electron-microscopical characteristics of isolated urinary bladders from toads (Bufo bufo) maintained in either tap water or 0.7% saline (0.7 g NaCl in 100 ml. H(2)O) for 10 days to 2 months.2. In the absence of Pitressin, isolated bladders from saline-adapted toads showed:(a) markedly, and significantly, lower osmotic water flow;(b) moderately, but not significantly, lower urea permeability;(c) no significant change in net sodium transport (measured as short-circuit current, I(sc)); and(d) significantly smaller intercellular space/mucosal cell ratios in electron-micrographs.3. Differences in the transport and electron-microscopical characteristics between bladders from water-exposed and saline-adapted toads became more evident in the presence of exogenous Pitressin (10 m-u./ml. serosal solution):(a) the stimulating influence of Pitressin on osmotic water flow, short-circuit current and urea permeability was considerably smaller in bladders from saline-adapted toads than in those from water-exposed toads;(b) the influence of Pitressin on short-circuit current was reduced more profoundly than that on either water flow or urea permeability;(c) the Pitressin-induced increment in intercellular space/mucosal cell ratio was significantly smaller in electron-micrographs of bladders from saline-adapted toads than in those from water exposed toads.4. The effects of saline adaptation are discussed in relation to decreased permeability of mucosal membrane barriers.

Animals↗
PubMed1994

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

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.

Amiloride↗
PubMed2005

The interplay of cutaneous water loss, gas exchange and blood flow in the toad, Bufo woodhousei: adaptations in a terrestrially adapted amphibian.

Toads experiencing dehydrating conditions exhibit complex physiological and behavioral responses, some of which can potentially impact cutaneous gas exchange, an important component of total gas exchange. We measured the effect of dehydration on cutaneous gas exchange in the xeric-adapted toad Bufo woodhousei. First, two pharmacological agents were used to stimulate cutaneous blood flow--phentolamine (an alpha-blocker) and isoproterenol, a beta-stimulant and powerful cardio-accelerator--to determine a relationship between cutaneous blood flow and water loss. Both drugs increased heart rate and blood pressure, and caused visually evident extensive vasodilation of the skin. Untreated toads in a dry air stream took an average of 10.1+/-0.7 h to dehydrate to 80% body mass, while animals treated with isoproterenol and phentolamine requires only 7.2+/-0.8 h and 7.4+/-0.9 h, respectively. Rehydration, which was more rapid than dehydration, was similarly accelerated in pharmacologically treated toads. Cutaneous gas exchange (M(O2), M(CO2)) and C18O diffusing capacity (D(Skin)C18O) were then examined in unanesthetized toads under different states of body hydration. Blood gases and hematocrit were measured separately but under identical conditions. In fully hydrated toads at 23-25 degrees C, cutaneous gas exchange values were: M(O2) = 1.43+/-0.47 micromol g(-1) h(-1), M(CO2) = 1.75+/-0.85 micromol g(-1) h(-1), and the respiratory exchange ratio R = 1.36+/-0.56 (N=6, mean + 1 S.D.). D(Skin)C18O was 0.48+/-0.03 micromol g body mass(-1) h(-1) kPa. Following an enforced 20-25% loss of body water, D(Skin)C18O fell by nearly 50% to 0.28+/-0.09 micromol g(-1) h(-1) kPa. However, cutaneous M(O2), M(CO2) and R were unchanged at 1.48+/-0.15 micromol g(-1) h(-1), 1.72+/-0.29 micromol g(-1) h(-1) and 1.13+/-0.08 micromol g(-1) h(-1), respectively. Partial pressure of arterial (sciatic) oxygen, Pa(O2), normally about 12-13 kPa, remained unchanged by dehydration, but Pa(CO2) increased about 250% from 0.93+/-0.27 up to 2.27+/-0.93 kPa. The fall in D(Skin)C18O during dehydration presumably results at least in part from decreased cutaneous blood flow, possibly in an attempt to reduce the transcutaneous water loss that would otherwise result during dehydrating conditions. Concurrently, cutaneous M(CO2) is maintained under dehyrdating conditions by a greatly increased Pa(CO2) diffusion gradient across the skin. Thus, Bufo woodhousei appears able to restrict cutaneous blood flow without compromising vital cutaneous CO2 loss.

Adaptation, Physiological↗
PubMed1996

Characterization of the atrial natriuretic factor system in lungs of the toad Bufo paracnemis.

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, high-performance 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.

Amino Acid Sequence↗
PubMed2002

Discrete electrolytic lesion of the preoptic area prevents LPS-induced behavioral fever in toads.

The preoptic area (POA) plays an important role in fever in mammals, but the role of this region in fever in ectothermic vertebrates has never been assessed. Toads, like all ectotherms, regulate their body temperature (T(b)) primarily by behavior and develop behavioral fever when injected with lipopolysaccharide (LPS). Therefore, we tested the hypothesis that the POA plays a role in the behavioral fever induced by LPS in the toad Bufo paracnemis. We made electrolytic lesions in the POA of toads (0.3 mA, 8 s) and measured preferred T(b) using a thermal gradient. After a period of 24h inside the gradient chamber, control, sham-operated and lesioned toads were systemically injected with LPS (200 micro g kg(-1)) or pyrogen-free saline. There was no significant effect of POA lesion in animals at their normal preferred T(b). LPS caused a significant increase in preferred T(b) of control and sham-operated toads, but lesions in the POA abolished this response. These results indicate that the POA is an important site in the central nervous system of toads, and perhaps of all vertebrates, involved in the development of fever.

Animals↗
PubMed1993

Stimulation of phosphoinositides by agents that stimulate proton secretion in toad urinary bladder.

The urinary bladder of Bufo marinus excretes H+ and this excretion is increased by metabolic acidosis (MA), insulin (IN), prostaglandin E2 (PGE2), increases CO2, and aldosterone. The purpose of this experiment was to determine whether MA, IN, PGE2, CO2, and aldosterone stimulate inositol phosphate's (IP) formation in isolated cells of toad urinary bladder. Cells were prepared by treating bladder sacs with collagenase. Cells were obtained from 10 toads in MA and 10 normal toads, suspended in 2 ml of Ringer's solution containing LiCl (10 mM), myo-inositol (5 mM), and [3H]myo-inositol (10 microCi), and then incubated for 2 hr at 25 degrees C. Cells were homogenized and the IP fractions quantitated by column chromatography and liquid scintillation counting. The results were expressed as dpm (mu MPO4)-1 (hr)-1. The IP in MA cells was 44,202 +/- 4,646 and in normal toad cells it was 31,637 +/- 3,613 (P < 0.05). In a separate experiment, cells from 10 paired hemibladders were isolated from normal toads. The cells were treated exactly as above except there were no LiCl in the bath. LiCl was added to all baths after 2 hr and the experimental cells were challenged with IN, PGE2, increases CO2, and aldosterone for 20 min. The IP were quantitated as above. IN treatment stimulated inositol bisphosphate and inositol triphosphate (P < 0.01). PGE2 and increases CO2 also stimulated inositol triphosphate (P < 0.05). Aldosterone did not alter formation of any of the IP fractions. We conclude that MA, IN, PGE2, and increases CO2 stimulate IP formation in cells of toad urinary bladder and inositol triphosphate may be an important second messenger in mediating the response of MA, IN, PGE2, and increases CO2.

Acidosis↗
PubMed2002

Observations on myiasis by the calliphorid, Bufolucilia silvarum, in the eastern American toad (Bufo americanus americanus) from southeastern Wisconsin.

Larvae of certain species of blowflies (Calliphoridae) cause myiasis in amphibians which may result in significant mortality, yet there are few reports from North America. In this study, we observed primary myiasis in a population of juvenile eastern American toads (Bufo americanus americanus) collected during May-July 1998 from southeastern Wisconsin (USA). Nine (6%) of 140 toads were infected by the green blow fly (Bufolucilia silvarum) with a mean intensity of 10.5 +/- 7.2 (range = 1-24). Weekly parasite prevalence and mean intensity remained low, ranging from 0-20% and 2 +/- 1.4 to 14 +/- 6, respectively. We found: 1) flies lay eggs on healthy toads, 2) eggs hatch with first instar maggots penetrating under the skin, 3) maggots develop to mature third instars within 5-7 days, 4) maggots leave the host and form pupa within 8-11 days of hatching, and 5) maggots pupate within 7-9 days at room temperature. All infected toads died within 1-2 wk as a result of the infection. The low prevalence observed in this study and other reports of this species from mammalian and bird carcasses indicated that B. silvarum is probably a facultative parasite of toads and other amphibians in the United States. This is the first report of B. silvarum causing myiasis in Wisconsin amphibians and the first report in eastern American toads in the United States.

Animals↗
PubMed1984

Effect of oestradiol dipropionate and testosterone propionate on protein, RNA and DNA contents and RNase and DNase activity in the liver of the toad (Bufo melanosticus).

A single injection of oestradiol dipropionate increased the HSI and protein, RNA and DNA contents and decreased the RNase and DNase activities of the liver of male and female toads. The minimum effective dose of oestrogen required to induce most of these changes was found to be 1 microgram/g (single injection), but the liver RNA content increased at the dose of 0.5 microgram/g. Oestrogen in a dose of 0.1 microgram/g did not cause any of these changes in male and female toads. Testosterone propionate (0.1, 0.5, 1 or 2 micrograms/g, single injection) was mostly ineffective in these respects, while in male toads higher doses of testosterone (1 and 2 micrograms/g) enhanced the liver RNA content only. The oestrogenic responses occurred earlier in female toads than in males. The liver protein and DNA contents increased from the 3rd day in female and on the 5th day in male toads. The liver RNA reached the higher level from the 2nd day in female and from the 3rd day in male. The RNase and DNase activities were reduced from the 2nd and 3rd day, respectively, in female and on the 5th day in male toads.

Animals↗
PubMed1983

Acid-base metabolism, intracellular pH and water transport by the toad bladder.

A decrease in extracellular pH is well known to inhibit vasopressin stimulated water flow in the toad bladder. It remains unclear whether this inhibition is the result of the effect of extracellular pH per se or the consequence of altered intracellular pH. In the present study we evaluated the effect of several maneuvers capable of altering intracellular pH on vasopressin or cyclic AMP stimulated water flow in the toad bladder in the absence of alterations of extracellular pH. In the presence of a normal extracellular pH, bladders subjected to a high partial pressure of CO2 or bladders from acidotic toads had a significant decrease in vasopressin or cyclic AMP stimulated water flow as compared to controls. We also examined the effect of maneuvers capable of increasing intracellular pH on vasopressin and cyclic AMP stimulated water flow. Intracellular alkalosis was induced by exposing the bladders in vitro to NH4Cl at pH 8 or to acetazolamide. Both maneuvers resulted in a significant decrease in vasopressin, but not in cyclic AMP stimulated water flow. Bladders removed from alkalotic toads, incubated in a normal extracellular pH also showed a decrease in AVP stimulated water flow. Intracellular muscle pH assessed with phosphorus nuclear magnetic resonance, was not different among bladders from control, acidotic and alkalotic toads. It is concluded that alterations of intracellular pH, in the absence of alterations of extracellular pH, are important in regulation of water transport in the toad bladder in response to vasopressin or cyclic AMP. In addition, metabolic acidosis or alkalosis alters AVP or cyclic AMP stimulated water flow by a mechanism independent of the intracellular pH.

Acetazolamide↗
PubMed2001

Seasonal changes in the preferred body temperature, cardiovascular, and respiratory responses to hypoxia in the toad, Bufo paracnemis.

Estivation is accompanied by a reduction of oxygen consumption in amphibians during drought. We tested the hypothesis that, during the dry season, the toad Bufo paracnemis selects a lower preferred body temperature (T(b)), and would be less sensitive to hypoxia, than during its active period. Therefore, during winter (dry season in São Paulo state, Brazil) and summer, we measured the effects of hypoxia (7% inspired O(2)) on preferred T(b). Additionally, pulmonary ventilation, heart rate, blood pressure, and oxygen consumption were also measured in toads at 15 and 25 degrees C. Blood gases were measured at 25 degrees C. Oxygen consumption was significantly higher during summer in toads at 25 degrees C. Under normoxia, preferred T(b) was higher during summer than during winter, and hypoxia caused a drop in preferred T(b) during both seasons. In both seasons, toads at 15 degrees C showed reduced pulmonary ventilation, heart rate, and blood pressure, and hypoxia had no effect. At 25 degrees C during summer only, hypoxia caused an increase in ventilation. Season had no effect on blood gases. We conclude that B. paracnemis displays an endogenous seasonal pattern of thermoregulation and control of ventilation. The decreased preferred T(b) and the physiological responses to hypoxia may be beneficial to toads encountering drought and when food is not available.

Adaptation, Physiological↗
PubMed2004

Seasonal effects of dehydration on glucose mobilization in freeze-tolerant chorus frogs (Pseudacris triseriata) and freeze-intolerant toads (Bufo woodhousii and B. cognatus).

It has been hypothesized that freeze-tolerance in anurans evolved from a predisposition for dehydration tolerance. To test this hypothesis, we dehydrated summer/fall-collected and winter acclimated freeze-tolerant chorus frogs and dehydration-tolerant, but freeze-intolerant, Woodhouse's and Great Plains toads to 25% and 50% body water loss (BWL). Following treatments, we measured glucose, glycogen, and glycogen phosphorylase and glycogen synthetase (summer/fall only) activities in liver and leg muscle. Hepatic glucose levels were not significantly altered by dehydration in either summer/fall-collected frogs or toads. Conversely, winter acclimated frogs did show an increment (2.9-fold) in hepatic glucose with dehydration, accompanied by a reduction in hepatic glycogen levels. Winter acclimated toads did not mobilize hepatic glucose in response to dehydration. Further, hepatic glycogen and phosphorylase activities did not vary in any consistent manner with dehydration in winter toads. Mean leg muscle glucose values were elevated at 50% BWL relative to other treatments, significantly so compared to 25% BWL for summer/fall-collected frogs. The pattern of hepatic glucose mobilization with dehydration in winter frogs is consistent with that in other freeze-tolerant frog species, and provides additional support for the hypothesis that freezing tolerance evolved from a capacity for dehydration tolerance. However, the lack of hepatic glucose mobilization in response to dehydration in fall frogs suggests that a seasonal component to dehydration-induced regulation of glucose metabolism exists in chorus frogs. Furthermore, the absence of a dehydration-induced mobilization of hepatic glucose at both seasons in toads suggests that this dehydration response is not universal for terrestrial anurans.

Acclimatization↗
PubMed2004

Effects of exercise on plasma catecholamine levels in the toad, Bufo paracnemis: role of the adrenals and neural control.

Resting plasma epinephrine (E) and norepinephrine (N) concentrations for intact toads (Bufo paracnemis) were 5.57+/-1.0 and 0.88+/-0.38 ng/ml, respectively. Exercise induced a significant increase in heart rate, blood pressure and plasma epinephrine (about 4.3 times), whereas norepinephrine remained unchanged. The resting [E]/[N] ratio was 6.3 and increased to 32.9 during exercise. Adrenal denervation did not alter the basal plasma catecholamine or norepinephrine levels after exercise, but prevented the increase in epinephrine during exercise, suggesting that in the intact toad this increase is due to adrenal secretion whereas resting norepinephrine may be liberated by extra-adrenal chromaffin tissues. This also suggests that the adrenal glands can release selectively the two catecholamines. The increases in heart rate and blood pressure in denervated toads were not significantly different from those of intact animals, suggesting that during exercise the sympathetic nerves play the main role in inducing cardiovascular responses. Spinal transection induced a significant increase in basal norepinephrine levels, which remained elevated after exercise. Since spinal toads are unable to perform spontaneous movements it is possible that this increase may be caused by this stressful condition. The increases in heart rate and blood pressure observed in spinal toads during exercise may be due to direct mechanical effects of venous return on the heart.

Adrenal Glands↗
PubMed1984

Anion permeability of toad skeletal muscle incubated in plasma and ringer solution.

Isolated semitendinosus muscles of the toad were incubated in both Ringer solution and toad plasma for periods ranging from 45 to 170 minutes. The extracellular volume (ECV), as measured from the equilibrium distribution of 35SO4(2-), was found to increase (in gram extracellular (EC) water/gram wet weight) from 0.15 +/- 0.01 to 0.20 +/- 0.02 in muscles incubated in Ringer solution, whereas the ECV was found to remain constant in tissues incubated in plasma (0.16 +/- 0.01 to 0.17 +/- 0.01), for periods up to 170 minutes. Since the true ECV, as measured morphometrically, was found to remain unchanged for muscles incubated in Ringer solution for up to 3 hours, the data for these muscles were interpreted to represent a change in anion permeability of the cellular membranes in response to the absence of some plasma fraction from the Ringer solution (with an electrolyte composition and pH similar to that of plasma). When toad plasma albumin was added to the Ringer solution, in physiological concentrations, the ECV of the muscles was observed to remain relatively constant during the 3-hour incubation period. This albumin effect on anion permeability was found to be albumin concentration-dependent. Further, other sources of albumin were found to have varying degrees of effect on the anion permeability of toad muscle, with bovine albumin serving as the most acceptable substitute for toad albumin, when incubating semitendinosus muscles.

Animals↗
PubMed2001

Non-invasive treatments of luteinizing hormone-releasing hormone for inducing spermiation in American (Bufo americanus) and Gulf Coast (Bufo valliceps) toads.

As many as 20% of all assessed amphibian species are threatened with extinction, and captive breeding programs are becoming important components of conservation strategies for this taxon. For some species, exogenous hormone administration has been integrated into breeding protocols to improve propagation. However, most treatments are administered by an intraperitoneal injection that can be associated with some risks. The general goal of this study was to identify a non-invasive method of applying luteinizing hormone-releasing hormone (LHRH), which reliably induces sperm release in toads. Specific objectives were to 1) test the spermiation response after topical application of different LHRH doses to the abdominal seat region, 2) evaluate the effects of adding the absorption enhancers dimethyl sulfoxide (DMSO), acetone, and glyceryl monocaprylate (GMC) to the LHRH, 3) assess the spermiation response after oral delivery of LHRH in a mealworm vehicle, and 4) compare sperm characteristics and spermiation responses to treatments in two different toad species. Male American (n = 9) and Gulf Coast (n = 7) toads were rotated systematically through a series of treatments. Urine was collected and evaluated for the presence of sperm at 0, 3, 7, 12, and 24 hours post-treatment. There were no statistical differences in spermiation induction or sperm characteristics between American and Gulf Coast toads after the treatments. Oral administration of 100 &mgr;g LHRH was occasionally successful in inducing spermiation, but results appeared largely unreliable. Ventral dermal application of 100 or 10 &mgr;g LHRH in 40% DMSO were more effective (P < 0.05) at inducing spermiation compared with the other treatments tested, eliciting sperm release in more than 70% of toads tested. In breeding programs for rare and/or fragile anurans, these non-invasive methods of exogenous hormone administration might be preferred over intraperitoneal injections. Zoo Biol 20:63-74, 2001. Copyright 2001 Wiley-Liss, Inc.

Journal Article↗
PubMed2001

Effects of ozone on evaporative water loss and thermoregulatory behavior of marine toads (Bufo marinus).

Ozone (O(3)) is a strong pulmonary irritant and causes a suite of respiratory tract inflammatory responses in humans and other mammals. In addition to lung injury, rodents exposed to O(3) exhibit a pronounced decrease in core body temperature at rest, which may offer a protective effect against O(3) damage. The effects of O(3) on other vertebrates have not been studied. Compared to individuals exposed to air (N=34), Bufo marinus toads exposed to O(3) (N=32) for 4 h lost 3.78 g body mass (adjusted mean from analysis of covariance, body mass mean+/-SD, 90.1+/-21.90 g). We tested the thermoregulatory responses of 22 toads in a thermal gradient 1, 24, and 48 h after 4-h exposure to air (N=11) or 0.8 ppm O(3) (N=11). Individual toad thermal preferences were also significantly repeatable across all trials (intraclass correlation=0.66, P <0.001). We did not observe a direct effect of O(3) exposure on the preferred body temperatures (PBT) of toads. However, O(3) exposure did have an indirect effect on selected temperatures. Ozone-exposed toads with higher evaporative water loss rates, in turn, also selected lower PBT, voluntary minimum, and voluntary maximum temperatures 24 h post-exposure. Ozone exposure may thus alter both water balance and thermal preferences in anuran amphibians.

Administration, Inhalation↗
PubMed1995

Circulating catecholamine and glucose concentrations in Japanese toads (Bufo japonicus) during the breeding season.

We investigated the relationship between catecholamine neurohormones and glucose during seasonal reproductive activity in Japanese toads (Bufo japonicus). Field studies found that plasma epinephrine concentration increased as toads migrated to their breeding ponds, where amplexus most frequently took place. Blood glucose concentration also increased as toads arrived at the ponds, even though these animals did not eat during the breeding season, and there was a positive correlation between epinephrine and glucose levels. Blood glucose concentration was higher in amplectic than in solitary males, whereas this relationship did not occur in females. For both males and females, plasma epinephrine concentration was elevated during amplexus. The plasma concentration of norepinephrine was lower than that of epinephrine and did not correlate with either the proximity of the animal to the breeding ponds or the blood glucose concentration. Laboratory experiments showed that systemic injection of [Trp7,Leu8]gonadotropin-releasing hormone (sGnRH) increased plasma epinephrine to levels characteristic of amplectic feral toads. These results suggest that a physiological role of GnRH-like peptides may be to stimulate epinephrine secretion and consequently to increase glucose production in toads under the starvation conditions associated with the breeding migration.

Animals↗
PubMed1996

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.

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 microM 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 microM. In toad fibres, 5 microM 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 microM 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 microM 2,5-di-(tert-butyl)-1, 4-hydroquinone. In the rat fibres, 5 and 20 microM 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 Ca(2+)-dependent damage) and the sarcoplasmic reticulum was reloaded with Ca2+. In the toad fibres, 5 microM 2,5-di-(tert-butyl)-1, 4-hydroquinone had a similar effect on depolarisation-induced force responses to that observed at 20 microM 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.

Animals↗
PubMed1993

Comparative enzymology of AMP deaminase, adenylate kinase, and creatine kinase in vertebrate heart and skeletal muscle: the characteristic AMP deaminase levels of skeletal versus cardiac muscle are reversed in the North American toad.

The specific activity of three characteristic enzymes, adenylate deaminase, adenylate kinase, and creatine kinase, in the skeletal muscles and heart of a variety of vertebrate land animals, including the human, are surveyed. Data from this study and available studies in the literature suggest that adenosine monophosphate deaminase in land vertebrates is quite high in white skeletal muscle, usually somewhat lower in red muscle, and 15- to 500-fold lower in cardiac muscle. Adenosine monophosphate deaminase is active primarily under ischemic or hypoxic conditions which occur frequently in white muscle, only occasionally in red muscle, and ought never occur in heart muscle, and this may therefore account for observed enzyme levels. The common North American toad, Bufo americanus, provides a striking exception to the rule with cardiac adenosine monophosphate deaminase as high as in mammalian skeletal muscle, whereas its skeletal muscle level of adenosine monophosphate deaminase is several times lower. The exceptional levels in the toad are not due to a change in substrate binding and are not accompanied by comparable change in the level of adenylate or creatine kinase. Nor do they signal any major change in isozyme composition, since a human muscle adenosine monophosphate deaminase-specific antiserum reacts with toad muscle adenosine monophosphate deaminase, but not with toad heart adenosine monophosphate deaminase. They do not represent any general anuran evolutionary strategy, since the bullfrog (Rana catesbeiana) and the giant tropic toad (Bufo marinus) have the usual vertebrate pattern of adenosine monophosphate deaminase distribution.(ABSTRACT TRUNCATED AT 250 WORDS)

AMP Deaminase↗