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[Hematologic values and altitudinal distribution of Chilean amphibians].

Blood values were measured on a series of Chilean species of frogs and toads from low and high altitudes, ranging from near sea level up to 4,500 m. Average values of red cell number, blood hemoglobin concentration and cell hemoglobin concentration were all higher in the high altitude group (6 species), while the average cell size was larger in the low altitude group (16 species). Hematocrits and cell hemoglobin content showed poor or no correlation with the altitudinal distribution of the examined species.

Altitude↗

Oviduct structure and function and reproductive modes in amphibians.

The structure and function of the oviducts of members of the three Orders of the Class Amphibia (Anura, frogs and toads; Urodela, salamanders and newts; Gymnophiona, caecilians) are well described for only a few species. Further, the majority of such descriptions relate only to temperate species that breed in water, lay their eggs there, and have free-living larvae, the presumed ancestral condition of oviparity. Many species of amphibians have derived reproductive modes. Such modes include breeding terrestrially and arboreally, making foam nests, parental transport of eggs and/or tadpoles, direct development (copulating on land, laying the eggs in terrestrial sites, fully metamorphosed juveniles hatching, obviating the free-living larval stage). Other derived modes are ovoviviparity (developing embryos retained in the oviducts, born at a diversity stages of development, no maternal nutrition in addition to yolk) and viviparity (oviductal retention of developing young, maternal nutrition after yolk is resorbed, young born as fully metamorphosed juveniles). The amphibian oviduct is regionally differentiated to secrete varying numbers of layers of material around each egg, which function in fertilization, etc.; it is responsive to endocrine output and environmental mediation during the reproductive cycle; and it maintains developing embryos in some members of all three orders, some with oviductal epithelial secretion of nutrients. However, little is known of the structure-function relationships of the oviduct in species with derived reproductive modes. A comparison of oviduct morphology, function, endocrinology, ecology and phylogeny in amphibians with diverse reproductive modes suggests a number of highly productive avenues of investigation.

Amphibians↗

The mitochondria-rich cell of frog skin as hormone-sensitive "shunt-path".

Further investigations about the role of the mitochondria-rich cell (MR cell) in hormone-mediated transport regulation in the epithelium of frog skin brought the following results: Unlike toad bladder, in frog skin the spontaneous potential difference cannot be reversed when Na transport is blocked. A similar situation is obtained when, in addition to transport-blockade, one applies a chemical gradient for chloride to the epithelium. Under these conditions we found that in the intact preparation as well as in the separated epithelium: (i) the reversed current (RC) is linearly related to the number of MR cells; (ii) RC is mainly carried by a passive, transcellular chloride flux inwards and (iii) RC is sensitive to nor-adrenaline (10(-7) M). The beta-blocker propranolol abolishes this effect. We propose that the MR cells are the sites of transepithelial shunt-path and that this chloride flux is transcellular. As it is hormone sensitive, it could be an important regulatory instrument for the regulation of overall salt transport (internal shorting).

Amiloride↗

Identification of two types of neurophysins in Xenopus laevis neurointermediate pituitary homologous to mammalian MSEL- and VLDV-neurophysins.

UNLABELLED: Xenopus laevis neurophysins have been purified from neurointermediate pituitaries through high-pressure reverse-phase liquid chromatography and their N-terminal amino acid sequences have been determined by microsequencing. Two types of neurophysins, corresponding to mammalian MSEL- and VLDV-neurophysins, have been distinguished. A strong homology exists between neurophysins of Xenopus (Pipidae), frog (Ranidae) and toad (Bufonidae). Xenopus MSEL-neurophysin, as frog MSEL-neurophysin, has a high molecular mass suggesting that the C-terminal domain of the vasotocin precursor is not processed in contrast to the two-step processing observed for mammalian vasopressin precursor. ABBREVIATIONS: Mammalian neurophysins are termed MSEL- and VLDV-neurophysins according to the nature of residues in positions 2, 3, 6 and 7 (one-letter symbols for amino acids).

Amino Acid Sequence↗

Juvenile amphibians do not avoid potentially lethal levels of urea on soil substrate.

We examined the effects of a forest fertilizer (urea) on newly metamorphosed terrestrial amphibians (Western toads, Bufo boreas; Cascades frogs, Rana cascadae; long-toed salamanders, Ambystoma macrodactylum; and roughskin newts, Taricha granulosa). We examined avoidance behavior of Western toads and Cascades frogs on both paper towel and soil substrates dosed with urea (control and 100 kg N/ha and an additional treatment of 50 kg N/ha for Western toads on soil substrate) and avoidance behavior of long-toed salamanders on soil substrate dosed with urea. We further examined the survival and feeding behavior of all four species exposed to urea on soil substrate (100 kg N/ha) for 5 d. Juvenile Western toads and Cascades frogs avoided paper towels dosed with urea but did not avoid urea-dosed soil substrate. However, Western toads and Cascades frogs both suffered significant mortality when exposed to urea on a soil substrate for 5 d. Furthermore, after adjusting for weight, we found that urea-exposed juvenile Western toads and Cascades frogs consumed significantly fewer prey items (crickets) compared with nonexposed control animals. Long-toed salamanders did not discriminate against soil substrate dosed with urea, and neither long-toed salamanders nor roughskin newts died or reduced prey consumption as a result of urea exposure. Juvenile amphibians may not be able to detect and avoid harmful levels of urea fertilizer on a natural substrate. Furthermore, anthropogenic stressors such as urea fertilizer can significantly reduce the survival and prey consumption of juvenile amphibians. These effects are important to consider in light of possible threats to the conservation status of many amphibian species.

Agriculture↗

Oxygen consumption of a terrestrial toad (Bufo viridis) and semi-aquatic frog (Rana ridibunda).

1. Oxygen consumption was measured before and after dehydration at different ambient temperatures (Ta) in the terrestrial toad Bufo viridis and the semi-aquatic frog Rana ridibunda. 2. The metabolic rates at Tas between 14 and 27 degrees C were almost the same for R. ridibunda and B. viridis. 3. The metabolic rate at higher Tas (between 27 and 36 degrees C) was higher in R. ridibunda than in B. viridis. This situation was found before and after dehydration. 4. A similar situation was found with CO2 production, which was higher at high Tas in R. ridibunda compared with B. viridis.

Animals↗

The effect of calcium withdrawal on the structure and function of the toad bladder.

Previous reports have indicated that calcium is necessary to support active sodium transport by the toad bladder, and may be required as well in the action of vasopressin on both toad bladder and frog skin. The structure and function of the toad bladder has been studied in the absence of calcium, and a reinterpretation of the previous findings now appears possible. When calcium is withdrawn from the bathing medium, epithelial cells detach from one another and eventually from their supporting tissue. The short-circuit current (the conventional means of determining active sodium transport) falls to zero, and vasopressin fails to exert its usual effect on short-circuit current and water permeability. However, employing an indirect method for the estimation of sodium transport (oxygen consumption), it is possible to show that vasopressin exerts its usual effect on Q(oo2) when sodium is present in the bathing medium. Hence, it appears that the epithelial cells maintain active sodium transport when calcium is rigorously excluded from the bathing medium, and continue to respond to vasopressin. The failure of conventional techniques to show this can be attributed to the structural alterations in the epithelial layer in the absence of calcium. These findings may provide a model for the physiologic action of calcium in epithelia such as the renal tubule.

Animals↗

Phosphorylated metabolites and effects of amphotericin B and ouabain on phosphorylation state in amphibian cornea.

The phosphorylated metabolite content of endothelial denuded frog corneas and toad corneal epithelial cells was measured. The extract of frog corneas contained adenine, uridine and guanine nucleotides, in addition to various glycolytic intermediates, orthophosphate (Pi) and creatine phosphate. The relationship between the phosphorylation state: PS = [ATP]/[ADP] X [Pi] and the rate of active ion transport was studied in the toad cornea. This was considered by measuring the effects of 10(-5) M-amphotericin B and 10(-2) M-ouabain on the adenine nucleotide and Pi content of toad epithelial cells since these drugs selectively stimulate and inhibit active ion transport and metabolic rates, respectively. In order to calculate PS values, we derived and validated a relationship for converting content in terms of nmol/mg protein into concentration (mM). Incubation with amphotericin B in six different cell suspensions resulted in decreases of the ATP and ADP concentrations from 2.3 to 1.2 mM and 0.7 and 0.4 mM, respectively. The Pi concentration increased more than the ATP decrease from 6.2 to 12.2 mM. The average of these individual changes in each of the suspensions resulted in a significant decrease of the calculated PS from 599 +/- 226 to 342 +/- 127 M-1. However, ouabain increased the ATP concentration from 2.3 to 2.6 mM and decreased the ADP and Pi concentrations from 0.7 to 0.3 mM and 6.2 to 5.4 mM, respectively. The average of these individual changes in each of the suspensions resulted in a significant increase of the PS from 599 +/- 226 to 2975 +/- 1639 M-1. These results support the current notion that the PS is the feedback signal synchronizing metabolic rates with the energetic requirements of active Na and K transport.

Adenine Nucleotides↗

Basic electrical properties of tight epithelia determined with a simple method.

A simple method for the routine mounting of epithelia which greatly reduces or eliminates faulty sealing at the edge is described. The values of potential and resistance observed in the frog skin and toad urinary bladder are comparable to those found with more complicated methods designed to minimize edge damage. In agreement with observations in other epithelia, a direct relationship between short circuit and conductance is observed for frog skin and toad urinary bladder, implying that the relationship is a general feature of tight epithelia. Furthermore, the high values of transepithelial resistance imply that the lower limit for the paracellular shunt pathway resistance should be reset upwards.

Amiloride↗

Survivorship patterns of larval amphibians exposed to low concentrations of atrazine.

Amphibians can be exposed to contaminants in nature by many routes, but perhaps the most likely route is agricultural runoff in amphibian breeding sites. This runoff results in high-level pulses of pesticides. For example, atrazine, the most widely used pesticide in the United States, can be present at several parts per million in agricultural runoff. However, pesticide levels are likely to remain in the environment at low levels for longer periods. Nevertheless, most studies designed to examine the impacts of contaminants are limited to short-term (approximately 4 days) tests conducted at relatively high concentrations. To investigate longer-term (approximately 30 days) exposure of amphibians to low pesticide levels, we exposed tadpoles of four species of frogs--spring peepers (Pseudacris crucifer), American toads (Bufo americanus, green frogs (Rana clamitans), and wood frogs (Rana sylvatica)--at early and late developmental stages to low concentrations of a commercial preparation of atrazine (3, 30, or 100 ppb; the U.S. Environmental Protection Agency drinking water standard is 3 ppb). We found counterintuitive patterns in rate of survivorship. Survival was significantly lower for all animals exposed to 3 ppb compared with either 30 or 100 ppb, except the late stages of B. americanus and R. sylvatica. These survival patterns highlight the importance of investigating the impacts of contaminants with realistic exposures and at various developmental stages. This may be particularly important for compounds that produce greater mortality at lower doses than higher doses, a pattern characteristic of many endocrine disruptors.

Animals↗

Effect of synthesis inhibitors of thromboxane A2 and prostaglandin E2 on the regulation of sodium and water.

The aim of this study was to evaluate the response to water deprivation in rats during development in the presence and in the absence of either benzylimidazole or acetaminophen (10 mg/kg of each), inhibitors of the synthesis of thromboxanes and other arachidonic acid metabolites. Whereas water deprivation induced an increment in urine osmolality both in control and vehicle-treated animals, this response was blocked by benzylimidazole and acetaminophen in the 5-day-old rats. No effect was observed in the older rats. Benzylimidazole also increased sodium excretion in the newborn rat. To assess the effect of benzylimidazole on epithelial cells deprived of the influence of hemodynamic factors, sodium transport and water flow were studied in the frog skin and in the toad bladder, respectively. In the frog skin epithelium, benzylimidazole (10(-4) M) inhibited the vasopressin-stimulated (100 mU/ml) sodium transport and in the toad bladder it decreased the vasopressin-stimulated (5 mU/ml) hydroosmotic flow. Our results suggest that thromboxanes are necessary for a full development of the response to vasopressin in sensitive epithelia. In the rat, thromboxanes and other arachidonic acid metabolites appear to play a role in the neonate but not in the older animal.

Acetaminophen↗

Isometric contractile properties and instantaneous stiffness of amphibian skeletal muscle in the temperature range from 0 to 20 degrees C.

The isometric contractile properties of frog (Rana pipiens) and toad (Bufo bufo) sartorii have been studied over the temperature range from 0 to 20 degrees C. The isometric twitch tension was found to vary considerably between these two species and between muscles in the same species. Between 0 and 4 degrees C there was very little change in maximum isometric twitch tension. Between 4 and 12 degrees C several muscles from frog or toad showed a potentiation of twitch tension whereas others showed a decline. Over this temperature range the toad sartorii consistently demonstrated a greater potentiation. By 12 degrees C a steady decline in twitch tension in both muscles was seen as the temperature range the toad sartorii consistently demonstrated a greater potentiation. By 12 degrees C a steady decline in twitch tension in both muscles was seen as the temperature approached 20 degrees C. The maximum isometric tetanic tension recorded between 18 and 20 degrees C increased fractionally to an average of 1.504 +/- 0.029 (n = 4) for frog sartorii and to 1.377 +/- 0.008 (n = 5) for toad sartorii. The time to peak twitch tension and the half-relaxation time decreased markedly with an increase in temperature. Moreover, the half-relaxation time was reduced by a greater proportion than the time to peak twitch tension. Measurements of instantaneous stiffness by controlled velocity releases from the plateau of isometric tetani revealed that the large increase in isometric tetanus tension as the muscle was warmed was not accompanied by a corresponding increase in the total number of active cross-bridges. The possibility that a decreased availability of intracellular Ca2+ ions at the contractile sites contributing to the fall of isometric twitch tension at elevated temperatures is discussed. The possibility exists that at elevated temperatures a change inthe intrinsic contractile ability of the muscle occurs which produces an increased tension per cross-bridge.

Action Potentials↗

Impact of ammonium nitrate on growth and survival of six European amphibians.

We conducted static experiments to assess the effects of ammonium nitrate fertilizer on embryos and larvae of six European amphibians: sharp-ribbed salamander (Pleurodeles waltl), Iberian painted frog (Discoglossus galganoi), western spadefoot toad (Pelobates cultripes), common toad (Bufo bufo), natterjack toad (Bufo calamita), and common tree frog (Hyla arborea). Embryos were exposed to different and environmentally relevant concentrations of ammonium nitrate (0 to 200 mg NO(3)(-)/L) for 15 days. Hatching took place during the experiments. H. arborea was extremely sensitive and had high mortality after 8 days of exposure even at the lower fertilizer levels. D. galganoi and B. bufo were also very sensitive and had significant mortality after 15 days of exposure. The rest of the species did not suffer lethal effects but suffered abnormalities or decreased growth at the highest fertilizer concentrations. Chemical fertilizers or manure could have contributed to the observed decrease of B. bufo and D. galganoi in agricultural areas of the Iberian Peninsula during recent years. H. arborea was the most sensitive species studied. The results of our study showed that environmentally relevant levels of ammonium nitrate can induce mortality and might affect population dynamics of this species in agricultural environments.

Amphibians↗

Proton pump activity is required for active uptake of chloride in isolated amphibian skin exposed to freshwater.

Net proton secretion and unidirectional chloride fluxes were measured in isolated skin of toads ( Bufo bufo) and frogs ( Rana esculenta) mounted in an Ussing chamber and exposed to a Ringer's solution on the serosal side and a freshwater-like solution (1-3 mM Cl(-)) on the external side. Active proton secretion was 34.2+/-2.0 pmol.cm(-2).s(-1) ( n=18) in frog skin, and 16.7+/-1.7 pmol.cm(-2).s(-1) ( n=10) in toad skin. Proton secretion by toad skin was dependent on the transepithelial potential ( V(T)), and an amiloride-insensitive short-circuit current was stimulated by exogenous CO(2)/HCO(3)(-), indicating the presence of a rheogenic proton pump. Cl(-) influx was 37.4+/-7.5 pmol.cm(-2).s(-1) ( n=14) in frog skin and 19.5+/-3.5 pmol.cm(-2).s(-1) ( n=11) in toad skin. In toad skin, the mean Cl(-) flux ratio was larger than expected for simple electro-diffusion. In 8 of 11 sets of paired skins, influx was greater than the efflux indicating active uptake of Cl(-). Cl(-) influx in toad skin was unaffected by large perturbations (100-150 mV) of V(T), which was accomplished by adding amiloride to the outer bath under open circuit conditions. A component of the Cl(-) efflux seemed to be dependent on V(T). 4,4'-Diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS; 0.3 mM or 1.3 mM) inhibited Cl(-) influx and, surprisingly, increased Cl(-) efflux in toad skin. Influx and efflux of Cl(-) in toad skin were highly dependent on the external [Cl(-)] in the freshwater range (0.1-4 mM). (36)Cl(-) influx decreased whereas the total Cl(-) efflux increased as a function of external [Cl(-)]. These data indicate the presence of a DIDS-sensitive, electroneutral carrier mechanism with an external binding site for Cl(-). Ethoxzolamide (100 micro M), an inhibitor of carbonic anhydrase, reduced proton secretion and Cl(-) influx in frog skin. Concanamycin A (0.1-10 micro M), a specific vacuolar-type proton pump (V-ATPase) inhibitor, significantly reduced proton secretion in frog skin. In addition, concanamycin A (1 micro M) significantly reduced Cl(-) influx in frog skin. We suggest that the active proton secretion and Cl(-) influx are coupled. We hypothesise that an apical V-ATPase is capable of energising active Cl(-) uptake in fresh water by creating a favourable gradient for an apical HCO(3)(-) exit in exchange for external Cl(-). The data also suggest that a carbonic anhydrase activity provides H(+) and HCO(3)(-) for apically co-expressed proton pumps and Cl(-)/HCO(3)(-) exchangers.

Animals↗

Effect of theophylline on the apical sodium and chloride permeabilities of amphibian skin.

1. The effects of theophylline (1 mmol/l) on the sodium transport (short-circuit current, Isc) and transepithelial conductance (Gtotal) through toad (Bufo viridis) and frog (Rana temporaria and Rana esculenta) skin were investigated. 2. In toad skin incubated with nitrate Ringer solution on the apical side, theophylline induced an increase in Isc similar to that in frog skin bathed with chloride Ringer solution. 3. The increase in Isc could be attributed to recruitment of sodium channels, without affecting the single-channel current. 4. Chloride-bathed toad skin responded to theophylline with a large increase in transepithelial conductance, in addition to the increased Isc. 5. Chloride replacement by nitrate eliminated the effect of theophylline on the conductance increase, but the Isc response was even larger. 6. The results are discussed in relation to the localization of the cellular chloride pathway to the mitochondria-rich cells.

Amphibians↗

Ca(2+)-blockable, poorly selective cation channels in the apical membrane of amphibian epithelia. UO2(2+) reveals two channel types.

This study deals with the effect of mucosal UO2(2+) on the Ca(2+)-blockable, poorly selective cation channels in the apical membrane of frog skin and toad urinary bladder. Our data show that UO2(2+) inhibits the Na+ currents through the amiloride-insensitive cation pathway and confirm a previously described stimulatory effect on the amiloride-blockade Na+ transport. Noise analysis of the Ca(2+)-blockable current demonstrates that the divalent also depresses the low-frequency Lorentzian (fc = 11.7 Hz) in the power density spectrum (PDS) and reveals the presence of high-frequency relaxation noise (fc = 58.5 Hz). The action of UO2(2+) is not reversed upon washout and is not accompanied by noise, typically induced by reversible blockers. The divalent merely depresses the plateau of the low-frequency Lorentzian, demonstrating a decrease in the number of conductive cation channels. Similarly, with mucosal K+ and Rb+, UO2(2+) also unmasks the high-frequency Lorentzian by depressing the noise from the slowly fluctuating cation channels (type S). In all experiments with mucosal Cs+, the PDS contains high-frequency relaxation noise (fc = 75.1 Hz in Rana temporaria, and 65.4 Hz in Rana ridibunda). An effect of UO2(2+) on the Cs+ currents and Lorentzian plateaus could not be demonstrated, suggesting that this monovalent cation does not pass through type S channels. Experiments with the urinary bladder revealed only a UO2(2+)-insensitive pathway permeable for Na+, K+, Rb+, and Cs+. We submit that in frog skin two cation-selective channels occur, distinguished by their spontaneous gating kinetics, their sensitivity to UO2(2+), and their permeability for Cs+. In toad urinary bladder, only one kind of cation-selective channel is observed, which resembles the UO2(2+)-insensitive channel in frog skin, with fast open-closed kinetics (type F).

Amiloride↗