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[Phylogenetic studies on some Japanese amphibia by means of immunoelectrophoresis. II. Relationships of some amphibians to Japanese pond frogs (author's transl)].

Since the Japanese pond frogs (Rana nigromaculata and R. brevipoda) are known to be very closely allied with each other in morphological, ecological, physiological or immunological characters, the phylogenetical relationships between the Japanese pond frogs and other 10 species of Japanese amphibians were investigated by means of immunoelectrophoretic analysis of liver extract. The results obtained are as follows: (1) There are conservative antigens which are commonly found in all species of Anura. (2) The Japanese pond frogs have specific antigens. (3) R. nigromaculata and R. brevidpoda are very closely allied with each other. (4) Four species of the genus Rana (R. rugosa, R. catesbeiana, R. ornativentris and R. japonica) are closely related to the Japanese pond frogs. (5) Two species of the genus Rhacophorus (Rh. arboreus and Rh. burergeri) are related to the Japanese pond frogs. (6) R. limnocharis is related to the Japanese pond frogs at the same extent as the genus Rhcophorus is. (7) Tow species of the suborder Procoela (Hyla arbored and Bufo bufo) are only partially related to the Japanese pond frogs. (8) Cynops pyrrhogaster pyrrhogaster of Urodera had only a few common antigens with the Japanese pond frogs.

Amphibians↗

A light microscope study of the distribution of muscle in the frog esophagus and stomach.

The present study reports light microscopical observations of the distribution of muscle in the esophagus and stomach of both the bull frog (Rana catesbeiana) and the African clawed frog (Xenopus laevis). The external muscle coat of the upper half of the esophagus in both species had several collagen coated bundles of striated muscle fibres around the circumference. These striated muscle bundles ran longitudinally from the pharynx to around the vicinity of the center of the esophagus. Beneath these striated muscle bundles was an inner circular layer of smooth muscle. In both species, the inner circular layer of smooth muscle was particularly thick in the region close to the pharynx. In the bull frog, the lower half of the esophagus lacked striated muscle. However, the circular smooth muscle layer, extending from the upper half of the esophagus, was also observed throughout the lower half of the esophagus. An outer longitudinal layer of smooth muscle developed towards the terminal portion of the esophagus such that in this region, both outer longitudinal and inner circular layers of smooth muscle were observed. Similarly in the African clawed frog, the inner circular layer of smooth muscle was continuous along the full length of the esophagus. Again, no striated muscle bundles were observed in the lower half of the esophagus. However, the outer longitudinal layer of smooth muscle was seen to develop in the middle region of the esophagus. Its muscle layer extended to the terminal portion of the esophagus. Thus, both outer longitudinal and inner circular layers of smooth muscle were observed throughout the lower half of the esophagus. In both frogs, the thickness of the outer longitudinal and inner circular layers of smooth muscle changed before and after the esophago-gastric junction. In both frogs, no muscularis mucosa was observed in the esophageal wall. However, in the lower half of the esophagus of the African clawed frog, small bundles of smooth muscle were observed here and there in the submucosa. A fully developed muscularis mucosa with both outer longitudinal and inner circular layers was observed in the upper stomach of both frogs.

Animals↗

[The Infection Status Of Sparganum And Gnathostoma In Frogs Of Southern Part Of Korea]

A survey on the infection status of sparganum and Gnathostoma in frogs was carried out in 11 areas in Gyeongsangnam-do and Cheonlanam-do province, a southern part of Korea. 1. Among 626 frogs examined, 4 percent were infected with sparganum, but none with Gnathostoma. The infection rates of sparganum were variable according to the areas. Those from Kimhae, Sunchun, Jinhae, Sachun, Kosung, Keoje, and Hadong were 16 %, 9.1 %, 9.1 %, 4.1 %, 2 %, 2 %, and 1.5 % respectively, but frogs from Hapchun, KwangJu, Namwon and Sanchung were not found infected. 2. Rana nigromaculata was the only species infected with sparganum. Rana rugosa and Bombina orientalis were not infected. 3. Sparganum was mainly found in the legs and the trunk of frog. About 92.5 % of worms were detected in the femoral intermuscular connective tissue. 4. Number per infected frog was in range of one to five, and 2.8 % of frogs were infected by only one worm. 5. The heavier the frog weight, the higher the infection rate of sparganum. In frogs of 51-60 grams, 28.6 % were infected.

Journal Article↗

Prevalence of skeletal and eye malformations in frogs from north-central United States: estimations based on collections from randomly selected sites.

Skeletal malformation rates for several frog species were determined in a set of randomly selected wetlands in the north-central USA over three consecutive years. In 1998, 62 sites yielded 389 metamorphic frogs, nine (2.3%) of which had skeletal or eye malformations. A subset of the original sites was surveyed in the following 2 yr. In 1999, 1,085 metamorphic frogs were collected from 36 sites and 17 (1.6%) had skeletal or eye malformations, while in 2000, examination of 1,131 metamorphs yielded 16 (1.4%) with skeletal or eye malformations. Hindlimb malformations predominated in all three years, but other abnormalities, involving forelimb, eye, and pelvis were also found. Northern leopard frogs (Rana pipiens) constituted the majority of collected metamorphs as well as most of the malformed specimens. However, malformations were also noted in mink frogs (R. septentrionalis), wood frogs (R. sylvatica), and gray tree frogs (Hyla spp.). The malformed specimens were found in clustered sites in all three years but the cluster locations were not the same in any year. The malformation rates reported here are higher than the 0.3% rate determined for metamorphic frogs collected from similar sites in Minnesota in the 1960s, and thus, appear to represent an elevation of an earlier baseline malformation rate.

Animals↗

Comparison of properties of Ca2+ release channels between rabbit and frog skeletal muscles.

Biochemical investigation of Ca2+ release channel proteins has been carried out mainly with rabbit skeletal muscles, while frog skeletal muscles have been preferentially used for physiological investigation of Ca2+ release. In this review, we compared the properties of ryanodine receptors (RyR), Ca2+ release channel protein, in skeletal muscles between rabbit and frog. While the Ryr1 isoform is the main RyR of rabbit skeletal muscles, two isoforms, alpha- and beta-RyR which are homologous to Ryr1 and Ryr3 isoforms in mammals, respectively, coexist as a homotetramer in a similar amount in frog skeletal muscles. The two isoforms in an isotonic medium show very similar property in [3H]ryanodine binding activity which is parallel to Ca2+ -induced Ca2+ release (CICR) activity, and make independent contributions to the activities of the sarcoplasmic reticulum. CICR and [3H]ryanodine binding activities of rabbit and frog are qualitatively similar in stimulation by Ca2+, adenine nucleotide and caffeine, however, they showed the following quantitative differences. First, rabbit RyR showed higher Ca2+ affinity than the frog. Second, rabbit RyR showed higher activity in the presence of Ca2+ alone with less stimulation by adenine nucleotide than the frog. Third, rabbit RyR displayed less enhancement of [3H]ryanodine binding by caffeine in spite of having a similar magnitude of Ca2+ sensitization than the frog, which may explain the occasional difficulty by researchers to demonstrate caffeine contracture with mammalian skeletal muscles. Finally, but not least, rabbit RyR still showed marked inhibition of [3H]ryanodine binding in the presence of high Ca2+ concentrations in the 1 M NaCl medium, while frog RyR showed disinhibition. Other matters relevant to Ca2+ release were also discussed.

Animals↗

Nitric oxide synthase activity reveals an asymmetrical organization of the frog habenulae during development: A histochemical and cytoarchitectonic study from tadpoles to the mature Rana esculenta, with notes on the pineal complex.

In the adult frog, structural asymmetry of the left dorsal habenula in respect to the right counterpart has been repeatedly documented in previous studies. In the present investigation, histochemical expression of beta-nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity was examined in the habenulae of the developing and adult Rana esculenta. In tadpoles and during metamorphosis, selective neuropil staining was consistently found within a lateral compartment of the medial subnucleus of the left dorsal habenula. The staining was still present in the same location, but much less intense, in the mature frog, indicating that the neurochemical pattern observed during development was at least in part transient. Thus, the present data point out a peculiar neurochemical pattern of the habenular asymmetry in the frog, suggesting that nitric oxide may be involved in the developmental shaping which leads to an asymmetrical configuration of the habenulae. In addition, NADPH-diaphorase-positive cells were detected in the frontal organ (the extracranial component of the pineal complex in strict relationship with the habenulae in the frog), and labeled fibers were found in the frontal nerve, which arises from the frontal organ. This latter finding supports the postulated relationship of the habenular asymmetry with the occurrence of the frontal organ. The finding of NADPH-diaphorase histochemical reactivity confined to a distinct portion of the medial subnucleus of the left dorsal habenula prompted a reexamination of the cytoarchitecture of the developing and mature habenular complex in the frog. The bicompartmentalization detected with histochemistry in the medial subnucleus of the left dorsal habenula of the developing and adult frog was fully supported by the study of Nissl-stained epithalamic sections. These data point out that the left-right structural differences of the frog habenular complex are more complex than previously believed, and may be subserved by chemically regulated developmental processes.

Animals↗

Interrelationship between food availability, fat body, and ovarian cycles in the frog, Rana tigrina, with a discussion on the role of fat body in anuran reproduction.

Long-term experiments were conducted to study the progression of vitellogenic cycles in Rana tigrina (an annual breeder) having different foraging backgrounds and held under conditions of weekly or daily food supply and in presence or absence of abdominal fat bodies. They were autopsied in June to assess fecundity. In nature an adult R. tigrina produces on an average 4,000 eggs/100 g body mass (b.m.) And spawns in June-July following monsoon rains. Weekly feeding from July to next breeding season, June resulted in a significant decrease in both fecundity (1700 eggs/100 g body b.m.) And mean size of eggs, compared to well-fed or wild-caught frogs. The abdominal fat bodies were barely seen in frogs fed weekly throughout, whereas in frogs fed weekly from July-December but daily from January onwards, the fat bodies became noticeable (1% of b.m.) And number and mean size of eggs increased significantly over those fed weekly throughout. Frogs captured in January possessed enlarged fat bodies (5% of b.m.), depicting a good foraging history. Maintenance of these frogs on a weekly feeding regimen led to an exhaustion of fat stores. They produced less number of eggs (2, 000/100 g b.m.) As compared to wild frogs but of normal size, whereas daily feeding slowed down a depletion of fat body mass and also significantly increased fecundity (3,000/100 g b.m.) Over the weekly fed individuals. Sham operation or fat body ablation in October or February had no significant effect on total fecundity per se (3,000-3,500 eggs/100 g b.m.) Compared to that of wild-caught frogs. However, eggs were significantly smaller due to fat body ablation despite daily feeding. The study shows that food abundance/fat bodies influence egg size and number in R. tigrina and that a direct or indirect functional relationship exists between fat body and ovarian cycles that are characteristically inverse to each other. J. Exp. Zool. 286:487-493, 2000.

Animals↗

Occurrence of bone fractures and parathyroid hyperplasia in paraphysectomized frogs (Rana catesbeiana).

The physiological role of the amphibian paraphysis was studied by removing the paraphysis from bullfrog tadpoles (Rana catesbeiana) and observing change in frog weight, bone development, and parathyroid glands 22 months later. Development was similar in control and paraphysectomized frogs until 6 months when the experimental frogs began to show greater weight gain which persisted for the remaining 16 months. At the time of sacrifice (22 months after paraphysectomy), the parathyroid glands of the experimental frogs were enlarged with a tenfold increase in wet weight and a sevenfold increase in dry weight. The glands from experimental frogs had numerous cysts filled with eosinophilic staining material; histologically the cell nuclei were rounded and the cytoplasm was vesiculated. At the time of sacrifice the experimental frogs had knoblike deformities along the shafts of the long bones. These deformities were due to callus formation around spontaneous bone fractures as revealed by X-rays of the tibia-fibulas and femurs. The dry weight was greater but calcium content less in femurs from experimental frogs. These findings indicate the paraphysis plays a role in calcium metabolism, possibly by producing a factor necessary for calcium action on cells.

Animals↗

Long-term survival of centrally projecting axons in the optic nerve of the frog following destruction of the retina.

A significant number of unmyelinated axons and their synaptic endings in the frog, Rana pipiens, were found to retain a normal morphology long after separation from their cell bodies. At the end of various survival periods following unilateral removal of the retina, horseradish peroxidase (HRP) was administered to the optic nerve stump by a fiber-filling method. In frogs maintained at 20 degrees C, unmyelinated optic nerve axons conducted HRP from the site of application in the orbit to layers A, C, and E of the contralateral optic tectum, even though their retinas had been removed up to 69 days earlier. Such fiber-filling was absent beyond 19 days in other frogs surviving at 35 degrees C. No labeled fibers were continuous with any intracerebral neurons. The HRP was always localized intraaxonally, and the marked axons and terminals were ultrastructurally normal. Counts of surviving axons from electron micrographs of the optic nerves showed that, at 20 degrees C, more than half of the normal complement of unmyelinated axons disappeared in the first 10 days. All the myelinated axons degenerated during the first 6 weeks survival. However, approximately 55,000 normal-appearing unmyelinated axons (12% of the unmyelinated fiber population) persisted in the optic nerve at 10 weeks following removal of the retina. The survival rate was lower at 35 degrees C. In other frogs, one eye was injected with 3H-leucine to initiate axonal transport into the retinal ganglion cell axons. That eye was removed 48 hours later. Autoradiographic analysis of brain sections of frog surviving an additional 31 to 61 days at 20 degrees C showed strong labeling of the optic tract and layers A, C, and E of the contralateral optic tectum. The absence of displaced ganglion cells that might exist within the optic nerve was verified by other observations. It is hypothesized that the potential shown by frog optic axons for long-term survival in the absence of the cell-body expresses a general property of vertebrate (and invertebrate) axons, rather than a special property of the frog optic nerve.

Animals↗

Differential localization of alkaline phosphatase in barrier tissues of the frog and rat nervous systems: a cytochemical and biochemical study.

We investigated the localization of alkaline phosphatase (AP) in the peripheral and central nervous systems of the frog (Rana pipiens) and rat. In the frog sciatic nerve, AP reaction product was seen as a precipitate within caveolae and vesicular profiles of perineurial cells, and frequently filled the extracellular space. In the rat peripheral nerve, AP reaction product appeared as small tufts on the cell surfaces and within vesicular profiles of endoneurial blood vessels. AP reaction product was not detected in the rat perineurium or in endoneurial blood vessels of the frog. In the frog central nervous system, AP reaction product was detected in the arachnoid membrane adjacent to the subarachnoid space, but not in the cerebral or pial vessels, whereas in the rat it was detected in the outer arachnoid membrane and in the cerebral and pial blood vessels. Biochemical analysis indicated a sevenfold higher AP activity in the frog perineurium over the endoneurium, whereas in the rat, threefold more activity was measured in the endoneurium over the perineurium. Levamisole, an AP inhibitor, decreased the enzyme activity by 95% in rat tissues, and by 70% in frog tissues and in plasma from both animals. Similar decrements were observed cytochemically. This study suggests that: (1) the distribution of AP varies between species, but that it is always present in at least one component of the blood-brain and blood-nerve barriers, (2) because barrier tissues of the nervous system have enzymatic activity, they may biochemically modify the adjacent environment, (3) vesicular profiles and caveolae in the blood vessels and perineurium may function as microenvironments for enzymatic activity, and (4) in the rat and frog, different isozymes of AP may be present.

Alkaline Phosphatase↗

Leukotrienes as mediators in frog virus 3-induced hepatitis in rats.

The role of leukotrienes was investigated in frog virus 3-induced hepatitis in rats. Frog virus 3 elicited an enhanced generation of cysteinyl leukotrienes in vivo as monitored by measurement of N-acetyl-leukotriene E4 as the major endogenous metabolite of cysteinyl leukotrienes secreted into rat bile. N-Acetyl-leukotriene E4 concentrations were elevated for more than 4 hr after frog virus 3 injection. In vitro experiments using cultured rat liver Kupffer cells of high purity indicated that these cells can produce and metabolize leukotrienes and are thus a possible source of leukotrienes elicited in vivo by frog virus 3. The selective 5-lipoxygenase inhibitor AA 861 and the dual inhibitor of arachidonate lipoxygenase and cyclooxygenase, BW 755C, reduced the hepatocellular injury after a high dose of frog virus 3 by about 50 and 80%, respectively, as judged from plasma activities of ALT and sorbitol dehydrogenase at 24 hr after frog virus 3 administration. Our in vivo and in vitro studies argue in favor of an important role of leukotrienes as mediators in frog virus 3 hepatitis in rats.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗

The influence of thyroxine and acclimation temperature on glycogen reserves of the frog Rana pipiens.

Leopard frogs were given daily injections of Na-l-thyroxine (2.0 mug/10 g body weight) for seven days, and control animals were given daily injections of the vehicle. During the period of treatment, half of the frogs were acclimated, in darkness and without food, at a constant temperature of 15 degrees C, whereas the remainder of the animals were acclimated at 25 degrees C. At the end of the seventh day, the frogs were killed by decapitation, and the glycogen concentration in samples of liver tissue and skeletal muscle was determined spectrophotometrically. Total hepatic glycogen of each frog subsequently was calculated from data on glycogen concentration and liver weight. Treatment of leopard frogs with thyroxine had no apparent effect on hepatic glycogen reserves of animals acclimated at 15 degrees C, but there was a striking reduction in glycogen content of livers from hyperthyroid frogs held at 25 degrees C. Mobilization of hepatic glycogen in thyroxine-treated animals held at 25 degrees C presumably was secondary to a general stimulation of oxidative metabolism. Treatment with thyroxine had no effect on the concentration of glycogen in skeletal muscle of leopard frogs held at either of the acclimation temperatures used in this study.

Acclimatization↗

Cooling rate influences cryoprotectant distribution and organ dehydration in freezing wood frogs.

Ice formation in the freeze-tolerant wood frog (Rana sylvatica) induces the production and distribution of the cryoprotectant, glucose. Concomitantly, organs undergo a beneficial dehydration which likely inhibits mechanical injury during freezing. Together, these physiological responses promote freezing survival when frogs are frozen under slow cooling regimes. Rapid cooling, however, is lethal. We tested the hypothesis that the injurious effects of rapid cooling stem from an inadequate distribution of glucose to tissues and an insufficient removal of water from tissues during freezing. Accordingly, we compared glucose and water contents of five organs (liver, heart, skeletal muscle, eye, brain) from wood frogs cooled slowly or rapidly during freezing to -2.5 degrees C. Glucose concentrations in organs from slowly cooled frogs were significantly elevated over unfrozen controls, but no significant increases occurred in rapidly cooled frogs. Organs from slowly cooled frogs contained significantly less water than did those from controls, whereas water contents from rapidly cooled frogs generally were unchanged. Rapid cooling therefore inhibited the production and distribution of cryoprotectant and organ dehydration during freezing. This inhibition may result from an accelerated, premature failure of the cardiovascular system.

Adaptation, Physiological↗

A study of ovarian follicular kinetics, oviduct, fat body, and liver mass cycles in laboratory-maintained Rana cyanophlyctis in comparison with wild-caught frogs.

Ovarian follicular dynamics and fluctuations in fat body, oviducal, and liver masses were studied in captive Rana cyanophlyctis in comparison with wild-caught frogs, sampled at monthly intervals over a period of 12 months. In both the captive and wild-caught frogs first growth phase (FGP) and second growth phase (SGP) or vitellogenic oocytes were produced throughout the period examined; however, changes in ovarian and oviducal weights were less marked in the former group. In the captive frogs SGP oocyte production was reduced by 50%, and, maximum ovarian weight and SGP oocyte number were attained 2-3 months earlier than in wild-caught controls. The FGP oocyte pool in laboratory-maintained frogs, however, was comparable with that of the corresponding wild-caught frogs. Captivity caused a threefold increase in atresia and reduced the number of oocytes reaching SGP. The depletion of fat stores in fat bodies during the later phases of captivity suggests that the deposition of lipids into oocytes (for SGP) was given priority over storage in the fat bodies. The low oviducal weights of captive frogs was correlated with a reduced number of SGP oocytes, which are the source of estrogen. On the other hand, liver weight remained high, indicating adequate hepatic vitellogenin synthesis. Possible reduction in its output was not detected, possibly due to the reduced number of follicles reaching SGP. The findings indicate that stress of captivity decreases gonadotrophins and estrogen levels. Oviducal growth is reduced in captive frogs.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Frog intestinal sac: a new in vitro method for the assessment of intestinal permeability.

The aim of this study was to evaluate a new experimental protocol utilizing isolated frog intestinal sacs for the assessment of intestinal drug permeability in humans. Segments of approximately 5.0 cm in length were used for these experiments. The intestinal sacs were filled with a solution of the appropriate drug in frog Ringer (FR) and immersed in a vial containing fresh FR. The transport was monitored for a period ranging from 2 to 5 h by moving the intestinal sac at each time point to a new vial containing fresh medium (Method A). Alternatively, according to Method B, at predetermined times aliquots of receiving mixture were taken up without removing the intestinal sac, and replaced with fresh drug-free FR. In all cases, the samples were analyzed by HPLC. A series of 20 noncongeneric drugs, predominantly absorbed by passive diffusion mechanism, was examined. The results indicate that drugs completely absorbed in humans had Papp values greater than 1 x 10(-6) cm/s, while drugs absorbed <90% had Papp values lower than 1 x 10(-6) cm/s. By plotting Log Papp values against percent human absorption, an approximately sigmoidal relationship was obtained. The frog intestinal sac method was evaluated as a permeability model to classify the 20 studied drugs into the Biopharmaceutics Classification System (BCS). By comparing the predictions made by this new approach with those reported in literature, it can be concluded that a satisfactory biopharmaceutical classification may be based on the Papp values determined by the frog intestinal permeability. Molecular properties relevant to passive intestinal permeability were considered to evaluate the correlation with the frog intestinal permeability rates recorded. Good relationships were observed when hydrogen-bonding parameters were expressed as a function of the Log Papp values. It can be concluded that the in vitro permeability coefficient deduced from isolated frog intestinal experiments can be used for predicting peroral absorption in humans for passively absorbed compounds. Computational methods for prediction of frog intestinal permeability may be applied in a highly simplified manner.

Animals↗

Glucocorticoids, androgens, testis mass, and the energetics of vocalization in breeding male frogs.

Male advertisement vocalization in frogs is known to be one of the energetically most expensive activities of ectothermic vertebrates. Glucocorticoids have marked effects on energy metabolism, and, generally, plasma concentrations of glucocorticoids increase during the course of prolonged exercise bouts. Androgen concentrations are also known to vary considerably among breeding male frogs. Intraspecific and interspecific comparisons were used to test for a relationship among androgen concentration, corticosterone concentration, testis mass, and the energetics of vocalization in natural populations of calling male frogs. The results of this study indicate that: (1) intraspecific variation in androgen and corticosteroid concentrations in breeding male frogs is positively correlated as a result of both interindividual variation in the amount of performed vocalization and the relationship between calling effort of an individual male and the level of calling in other males, (2) interspecific variation in corticosteroid concentration of calling male frogs is correlated with the relative energy expended in the species-specific vocalization, and (3) when differences in testis mass are controlled for, vocalization effort is correlated with androgen concentration among species of breeding male frogs. These findings are in contrast to some recent work reported from laboratory experiments on calling frogs.

Androgens↗

Potassium transport in red blood cells of frog Rana temporaria: demonstration of a K-Cl cotransport.

Pathways of K+ movement across the erythrocyte membrane of frog Rana temporaria were studied using 86Rb as a tracer. The K+ influx was significantly blocked by 0.1 mmol.l-1 ouabain (by 30%) and 1 mmol.l-1 furosemide (by 56%) in the red cells incubated in saline at physiological K+ concentration (2.7 mmol.l-1). Ouabain and furosemide had an additive effect on K+ transport in frog red cells. The ouabain-sensitive and furosemide-sensitive components of K+ influx saturated as f(K+)e with apparent Km values for external Ke+ concentration of 0.96 +/- 0.11 and 4.6 +/- 0.5 mmol.l-1 and Vmax of 0.89 +/- 0.04 and 2.8 +/- 0.4 mmol.l cells-1.h-1, respectively. The residual ouabain-furosemide-resistant component was also a saturable function of Ke+ medium concentration. Total K+ influx was significantly reduced when frog erythrocytes were incubated in NO3- medium. Furosemide did not affect K+ transport in frog red cells in NO3- media. At the same Ke+ concentration the ouabain-furosemide-insensitive K+ influx in Cl- medium was significantly greater than that in NO3- medium. We found no inhibitory effect of 1 mmol.l-1 furosemide on Na+ influx in frog red cells in Cl- medium. K+ loss from the frog erythrocytes in a K(+)-free medium was significantly reduced (mean 58%) after replacement of Cl- with NO3-. Furosemide (0.5 mmol.l-1) did not produce any significant reduction in the K+ loss in both media. The Cl(-)-dependent component of K+ loss from frog red cells was 5.7 +/- 1.2 mmol.l-1.h-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

A diazo-2 study of relaxation mechanisms in frog and barnacle muscle fibres: effects of pH, MgADP, and inorganic phosphate.

The aim of this study was to compare the effects of increased concentrations of MgADP, inorganic phosphate (Pi) and H+ ([MgADP], [Pi] and [H+], respectively) on the rate of relaxation in two different muscle types: skinned muscle fibres from the frog Rana temporaria and myofibrillar bundles from the giant Pacific acorn barnacle Balanus nubilus. Relaxation transients are produced by the photolysis of diazo-2 and are well fitted with a double exponential curve, giving two rate constants: k1 [5.6+/-0.1 s-1 for barnacle, n=30; 26.3+/-0.7 s-1 for frog, n=14 (mean+/-SEM)] and k2 [0.6+/-0.1 s-1 in barnacle, n=30; 10.4+/-1.0 s-1 in frog, n=14 (mean+/-SEM)], at 10 degrees C. Decreasing the pH by 0.5 pH units did not significantly affect k1 for barnacle relaxation [5.6+/-0.1 s-1 (mean+/-SEM), n=15] compared to the decrease in k1 of 40% seen in frog. Use of the Ca2+-sensitive fluorescent label acrylodan on barnacle wild-type troponin C demonstrated that decreasing the pH from 7.0 to 6.6 only alters the pCa50 value by 0.23 in the cuvette, while stopped-flow experiments with acrylodan revealed no significant change in koff from the labelled protein [322+/-32 s-1 at pH 7.0 and 381+/-24 s-1 (mean+/-SEM) at pH 6.6]. Increasing [MgADP] by 20 microM (50 microM added ADP) from control values of 50 microM in frog decreased k1 to 12.3+/-0.4 s-1 (mean+/-SEM, n=8), and at 400 microM MgADP, k1=9.6+/-0.1 s-1 (mean+/-SEM, n=12). In barnacle, 500 microM MgADP had a much smaller effect on k1 (4.0+/-0. 9 s-1, mean+/-SEM, n=8). Increasing the free [Pi] from the contaminant level of 0.36 mM to 1.9 mM slowed k1 by approximately 15% in barnacle [4.8+/-0.8 s-1, mean+/-SEM, n=7], compared to a approximately 30% reduction seen in frog. We conclude that the differences between barnacle and frog seen here are most probably due to different isomers of the contractile proteins, and that events underlying the crossbridge cycle are the same or similar. We interpret our results according to a model of crossbridge transitions during relaxation.

2-Naphthylamine↗