Viability of some metabolic processes in the isolated toad brain adapted to two osmotic environments.

The viability of the isolated toad brain in an aerated Ringer-like medium has been evaluated by the following criteria: 1) amino acid content before and after incubation; 2) accumulation of amino acids in the incubation medium; 3) a comparison of glucose utilization and [U-14C]glucose metabolism with that occurring in vivo; 4) tissue swelling; and 5) tissue lactate contents. On the basis of these criteria, the isolated toad brain, from toads adapted to a fresh-water or a salt-water environment, retains considerable metabolic integrity for at least 2 hr of incubation at 25 degrees C. Specifically, there was no swelling of the tissue, no apparent accumulation of lactate in the tissue, glucose appeared to be utilized at a rate not too different from that calculated for the toad brain in vivo, and the distribution of label from [U-14C]glucose had an overall pattern which resembled that observed in vivo. The tissue levels of amino acids were generally stable in vitro; however, there was a marked decline in the content of aspartate. The accumulation of amino acids in the medium varied considerably from one amino acid to another. Thus, there was very little net efflux of aspartate, GABA, and glutamate from the tissue but considerable net efflux of glutamine. This efflux of amino acids was greater from brains of hyperosmotically adapted toads than from the brains of toads adapted to fresh water by amounts proportional to their initial tissue contents.     

Trypsin affects basal and stimulated osmotic water permeability in isolated toad skin

1. We investigated the effect of trypsin (Tryp) on basal, stimulated and fluphenazine (FPZ)-inhibited net water flow (Jw) through isolated toad skin (Bufo arenarum). 2. Epidermal Tryp (20 min) promoted an increase in basal Jw which was dose-dependent (maximal with 0.5 mg/ml) and was prevented by a Tryp inhibitor (SBTI). 3. Tryp treatment inhibited the subsequent response to substances known to act before (oxytocin, Oxy) or after cyclic AMP (cAMP) generation (theophylline). 4. Tryp-induced Jw was not additive with the maximal response to Oxy or theophylline and did not modify FPZ's inhibitory effect on stimulated Jw. 5. Dermal Tryp (0.5 mg/ml, 20 min) did not modify basal, but inhibited Oxy and isoproterenol-stimulated Jw, without altering the response to theophylline or db-cAMP. 6. Collectively, our results show a differential action for epidermal and dermal Tryp. Tryp's side-selective action enables its use as a pharmacological tool in the functional dissection of Jw across toad skin.     

Very low osmotic water permeability and membrane fluidity in isolated toad bladder granules

Summary Osmotic water permeability of the apical membrane of toad urinary epithelium is increased greatly by vasopressin (VP) and is associated with exocytic addition of granules and aggrephores at the apical surface. To determine the physiological role of granule exocytosis, we measured the osmotic water permeability and membrane fluidity of isolated granules, surface membranes and microsomes prepared from toad bladder in the presence and absence of VP.P _f was measured by stopped-flow light scattering and membrane fluidity was examined by diphenylhexatriene (DPH) fluorescence anisotropy. In response to a 75mm inward sucrose gradient, granule size decreased with a single exponential time constant of 2.3±0.1 sec (sem, seven preparations, 23°C), corresponding to aP _f of 5×10^–4 cm/sec; the activation energy (E _a ) forP _f was 17.6±0.8 kcal/mole. Under the same conditions, the volume of surface membrane vesicles decreased biexponentially with time constants of 0.13 and 1.9 sec; the fast component comprised 70% of the signal. Granule, surface membrane and microsome time constants were unaffected by VP. However, in surface membranes, there was a small decrease (6±2%) in the fraction of surface membranes with fast time constant. DPH anisotropies were 0.253 (granules), 0.224 (surface membrane fluidity is remarkably lower than that of surface and microsomal membranes, and (4) rapid water transport occurs in surface membrane vesicles. The unique physical properties of the granule suggests that apical exocytic addition of granule membrane may be responsible for the low water permeability of the unstimulated apical membrane.     

Antidiuretic responses to osmotic, ionic or volume stimulation of the brain in the unanesthetized toad, Bufo arenarum Hensel

Single injections of 15 microliter of 1 M NaCl or 2M sucrose into the carotid system of normal unanesthetized toads induced a rapid and significant decrease of urine production. This appears to be the first report on the existence of a Verney-like phenomena in a non-mammalian vertebrate. This antidiuresis was blocked out in the hypophysectomized diencephalic lesioned animal. Concurrently, small volumes (4 microliters) of 1 M NaCl but not 2 M sucrose also induced antidiuresis when injected into the midbrain tegmentum of normal but not of hypophysectomized diencephalic lesioned toads. Larger volumes (6 microliters) of 2 M sucrose were needed to induce a similar antidiuresis in normals. Furthermore, even larger volumes (more than 8 microliters) of any of both solutions were able to induce oliguria in normal as well as in hypophysectomized toads. On the basis of these results, the following conclusion would be drawn: the brain of the toad is able to detect ionic and osmotic stimuli, these stimuli apparently affect different receptors in the brain, the antidiuresis initiated by these mechanisms is dependent on diencephalic integrity.     

Bioenergetic response of isolated nerve terminals of rat brain to osmotic swelling

The swelling of nerve terminals of rat brain in a hypotonic medium (230 mOsm) induced the potential-independent entrance of 45Ca2+ into synaptosomes and intrasynaptosomal mitochondria that changed the energy status of synaptosomes, the rate of O2 consumption and the content of ATP being decreased. The ratio ATP/ADP decreased from 6.5 +/- 0.26 (310 mOsm medium) to 3.1 +/- 0.18 (the medium 230 mOsm). Studies on the equilibrium distribution of K+ (86Rb+) and [3H]TPP+ showed that contents of these cations in the nerve terminals were virtually the same on incubation in both iso- and hypotonic media. This indicated that the swelling did not damage intrasynaptosomal mitochondria and plasma membranes of the synaptosomes. The inhibition of oxidative phosphorylation increased twofold the rate of glycolysis. The incubation of synaptosomes in calcium-free medium (230 mOsm) in the presence of EGTA (1 mM) prevented the inhibition of oxidative phosphorylation and synthesis of ATP by the osmotic swelling. Ruthenium Red (10 microM) in the medium 230 mOsm inhibited the entrance of 45Ca2+ into the intrasynaptosomal mitochondria and normalized the oxidative phosphorylation to the control level (310 mOsm medium). The decrease in the energy potential of synaptosomes induced by the hypoosmotic shock is suggested to be associated with the increase in Ca2+ content in the cytoplasm, its transport into the mitochondria, and the inhibitory effect on oxidative phosphorylation.     

Antidiuretic responses to osmotic cutaneous stimulation in the toad,Bufo arenarum

Summary Osmotic stimulation of the skin of the toadBufo arenarum with isotonic (115 mM) or hypertonic (400 mM) NaCl solutions produced a marked and reversible antidiuresis within 5 min. No changes in plasma osmolarity were detected in the course of this response.Hypophysectomized animals exhibited a lower and delayed antidiuresis when exposed to a hypertonic environment (400 mM NaCl). This antidiuretic response was drastically reduced in normal toads after ten consecutive days of administration of the sympatoplexic guanethidine.The existence of a feed-forward control of urine production initiated by cutaneous osmotic sensors and involving an adrenergic component is proposed.     

Capacitance,short-circuit current and osmotic water flow across different regions of the isolated toad skin

Summary The amphibian antidiuretic hormone, arginine vasotocin, stimulated osmotic water flow across isolated skin from the pelvic but not the pectoral skin of the toad, Bufo woodhouseii. Changes in the apical membrane capacitance were not observed for either region of the skin following treatment with arginine vasotocin when there was an osmotic gradient across the tissue. In the absence of an osmotic pressure gradient, the apical membrane capacitance of the pelvic skin increased from 2.8±0.5 to 3.3±0.6 F · cm^-2 after treatment with 5 · 10^-8 M arginine vasotocin. Under these conditions, apical membrane capacitance of the pectoral skin was 1.8±0.1 F · cm^-2 and did not change significantly after arginine vasotocin treatment. The amiloride-sensitive short-circuit current across the pelvic skin was stimulated by arginine vasotocin as was the density of channels in the apical membrane as determined by fluctuation analysis. Values for channel density in the pelvic skin also correlated with apical membrane capacitance and increased from 90 to 273 channels per m² of estimated membrane area following arginine vasotocin treatment. In the pectoral skin the stimulation of short-circuit current following arginine vasotocin treatment was small and an increase in channel density could not be demonstrated. The current through single Na⁺ channels in both regions of the skin did not different either before or after arginine vasotocin treatment.Abbreviations A amiloride - ADH antidiuretic hormone - AVT arginine vasotocin - C capacitance - C _a capacitance of apical membrane - f _c corner frequency - i single-channel current - osmotic water flow - IMP intramembrane particles - I _sc short-circuit current - amiloride-sensitive short-circuit current - M channel density - P _o probability of a channel being open - R channel receptor - R _a apical resistance - R _p paracellular resistance     

Inhibition of stimulated osmotic water flow by fluphenazine, a calmodulin inhibitor, in the isolated toad skin

The effects of fluphenazine, a phenothiazine calmodulin inhibitor, on the osmotic water flow (Posm) across isolated skins of Bufo arenarum toads were tested. Fluphenazine inhibited the increase in Posm induced by agents known to act through the cyclic AMP system (oxytocin, db-cAMP, theophylline, KCl and isoproterenol). The inhibitory effect was faster and more intense when the drug was present in the epidermal bath, and persisted after rinsing the preparation for 100 min. Our results indicate that phenothiazine's action may be primarily exerted at a site distal to cyclic AMP generation.     

Contribution of pH-sensitive metabolic processes to pH homeostasis in isolated rat kidney tubules

The metabolism of isolated rat kidney tubules suspended in calcium-free physiological saline buffered with phosphate was found to be sensitive to changes in the pH of the suspending medium. Lowering the pH from 7.8 to 6.4 brought about increases in the rates of oxidation of added succinate, glutamate or glutamine as well as in the production of glucose from lactate, glutamine, succinate and fructose. The cellular ATP level was also higher in tubules incubated at pH 6.4. In contrast, the utilization of added glucose was greater at pH 7.8 than at pH 6.4, a substantial amount of lactate being produced at the higher pH. When glucose and either lactate or glutamine were provided as co-substrates glucose was the preferred fuel at pH 7.8 but the alternative substrate was the more readily utilized at pH 6.4. As a consequence of the metabolic activities of the tubules the pH of the suspending medium changed, utilization of lactate, glutamate or glutamine causing a rise in pH while conversion of glucose to lactate caused a fall in pH. In cases where two substrates were metabolized concurrently over a period of 3 h the extracellular pH tended towards a plateau level of approximately pH 7.4. It is proposed that pH-sensitive metabolism in isolated kidney tubules contributes to pH homeostasis in the cellular environment.     

Solutes contributing to osmotic adjustment in cultured plant cells adapted to water stress

Osmotic adjustment was studied in cultured cells of tomato (Lycopersicon esculentum Mill cv VFNT-Cherry) adapted to different levels of external water potential ranging from −4 bar to −28 bar. The intracellular concentrations of reducing sugars, total free amino acids, proline, malate, citrate, quaternary ammonium compounds, K⁺, NO₃⁻, Na⁺, and Cl⁻ increased with decreasing external water potential. At any given level of adaptation, the maximum contribution to osmotic potential was from reducing sugars followed by potassium ions. The sucrose levels in the cells were 3- to 8-fold lower than reducing sugar levels and did not increase beyond those observed in cells adapted to −16 bar water potential. Concentrations of total free amino acids were 4- to 5-fold higher in adapted cells. Soluble protein levels declined in the adapted cell lines, but the total reduced nitrogen was not significantly different after adaptation. Uptake of nitrogen (as NH₄⁺ or NO₃⁻) from the media was similar for adapted and unadapted cells. Although the level of quaternary ammonium compounds was higher in the nonadapted cells than that of free proline, free proline increased as much as 500-fold compared to only a 2- to 3-fold increase observed for quaternary ammonium compounds. Although osmotic adjustment after adaptation was substantial (up to −36 bar), fresh weight (volume increase) was restricted by as much as 50% in the adapted cells. Altered metabolite partitioning was evidenced by an increase in the soluble sugars and soluble nitrogen in adapted cells which occurred at the expense of incorporation of sugar into cell walls and nitrogen into protein. Data indicate that the relative importance of a given solute to osmotic adjustment may change depending on the level of adaptation.     

Contribution of pH-sensitive metabolic processes to pH homeostasis in isolated rat kidney tubules.

The metabolism of isolated rat kidney tubules suspended in calcium-free physiological saline buffered with phosphate was found to be sensitive to changes in the pH of the suspending medium. Lowering the pH from 7.8 to 6.4 brought about increases in the rates of oxidation of added succinate, glutamate or glutamine as well as in the production of glucose from lactate, glutamine, succinate and fructose. The cellular ATP level was also higher in tubules incubated at pH 6.4 In contrast, the utilization of added glucose was greater at pH 7.8 than at pH 6.4, a substantial amount of lactate being produced at the higher pH. When glucose and either lactate or glutamine were provided as co-substrates glucose was the preferred fuel at pH 7.8 but the alternative substrate was the more readily utilized at pH 6.4. As a consequence of the metabolic activities of the tubules the pH of the suspending medium changed, utilization of lactate, glutamate or glutamine causing a rise in pH while conversion of glucose to lactate caused a fall in pH. In cases where two substrates were metabolized concurrently over a period of 3 h the extracellular pH tended towards a plateau level of approximately pH 7.4. It is proposed that pH-sensitive metabolism in isolated kidney tubules contributes to pH homeostasis in the cellular environment.     

Herbicidal effect on some metabolic processes of Azotobacter chroococcum

The effect of pyramin (1-phenyl-4-amino-5-chloro-piridazone-6) and venzar (3-cyclohexyl-5,6-trimethyleneuracil) on growth, oxygen uptake and nitrogenase activity was examined. The possibilities of herbicide accumulation in cells and binding by mucopolisaccharides were also tested. Stimulation of growth was observed in the presence of pyramin (20 and 100 ppm) but no influence of venzar was found. Venzar was found to penetrate into the cells. Pyramin did not penetrate into the cells but its content decreased in cultures of Azotobacter strains. However, no degradation products of pyramin were found. Pyramin was detected in mucopolisaccharide fractions in range 0.2-47 micrograms of Pyrazon per 100 mg. Pyramin and venzar reduced the acetylene reduction rate of crude extracts by 50% when doses of active substances were equal to the weight of crude extract protein contents. Doses required for similar effect in living cells were however more than fourteen times higher. Addition of polivinylpyrrolidone and albumin protected the N2-ase from negative effect of pyramin and to a lesser degree from that of venzar.     

Cellular changes in the toad urinary bladder in response to metabolic acidosis

Summary The urinary bladder ofBufo marinus excretes H⁺ and NH ₄ ⁺ , and the H⁺ excretion is increased when the animal is placed in metabolic acidosis. The mitochondriarich (MR) cells mediate the H⁺ excretion by the bladder. The purpose of this study was to determine if there is a change in MR cells of the bladder during metabolic acidosis. Bladders from normal toads and from toads that had been placed in metabolic acidosis were used. The bladders were mounted between plastic chambers and H⁺ excretion measured. The bladder was then fixed and prepared for scanning (SEM) and transmission (TEM) electron micrograph studies. SEM's at low magnification were used to count the various cell types and the TEM's were used to confirm the different cell types. Fields were randomly selected and a total of 2500 cells counted in each group. The bladders from toads in metabolic acidosis had a consistently higher ratio of MR cells to granular cells than did the normal bladders. These results indicate that during metabolic acidosis there is an increased number of MR cells in the bladder, and this increases the bladder's capacity to excrete H⁺.     

Ecological specialization in populations adapted to constant versus heterogeneous environments*

Populations vary in their degree of ecological specialization. An intuitive, but often untested, hypothesis is that populations evolving under greater environmental heterogeneity will evolve to be less specialized. How important is environmental heterogeneity in explaining among‐population variation in specialization? We assessed juvenile viability of 20 Drosophila melanogaster populations evolving under one of four regimes: (1) a salt‐enriched environment, (2) a cadmium‐enriched environment, (3) a temporally varying environment, and (4) a spatially varying environment. Juvenile viability was tested in both the original selective environments and a set of novel environments. In both the original and novel environments, populations from the constant cadmium regime had the lowest average viability and the highest variance in viability across environments but populations from the other three regimes were similar. Our results suggest that variation in specialization among these populations is most simply explained as a pleiotropic by‐product of adaptation to specific environments rather than resulting from a history of exposure to environmental heterogeneity.     

Activity,stability and flexibility in glycosidases adapted to extreme thermal environments

To elucidate the strategy of low temperature adaptation for a cold-adapted family 8 xylanase, the thermal and chemical stabilities, thermal inactivation, thermodependence of activity and conformational flexibility, as well as the thermodynamic basis of these processes, were compared with those of a thermophilic homolog. Differential scanning calorimetry, fluorescence monitoring of guanidine hydrochloride unfolding and fluorescence quenching were used, among other techniques, to show that the cold-adapted enzyme is characterized by a high activity at low temperatures, a poor stability and a high flexibility. In contrast, the thermophilic enzyme is shown to have a reduced low temperature activity, high stability and a reduced flexibility. These findings agree with the hypothesis that cold-adapted enzymes overcome the quandary imposed by low temperature environments via a global or local increase in the flexibility of their molecular edifice, with this in turn leading to a reduced stability. Analysis of the guanidine hydrochloride unfolding, as well as the thermodynamic parameters of irreversible thermal unfolding and thermal inactivation shows that the driving force for this denaturation and inactivation is a large entropy change while a low enthalpy change is implicated in the low temperature activity. A reduced number of salt-bridges are believed to be responsible for both these effects. Guanidine hydrochloride unfolding studies also indicate that both family 8 enzymes unfold via an intermediate prone to aggregation.     

Differential selection to avoid hybridization in two toad species

Abstract.— The fitness consequences of hybridization critically affect the speciation process. When hybridization is costly, selection favors the evolution of prezygotic isolating mechanisms (e.g., mating behaviors) that reduce heter-ospecific matings and, consequently, enhance reproductive isolation between species (a process termed reinforcement). If, however, selection to avoid hybridization differs between species, reinforcement may be impeded. Here, we examined both the frequency and fitness effects of hybridization between plains spadefoot toads ( Spea bombifrons ) and New Mexico spadefoot toads ( S. multiplicata ). Hybridization was most frequent in smaller breeding ponds that tend to be ephemeral, and heterospecific pairs consisted almost entirely of S. bombifrons females and S. multiplicata males. Moreover, in controlled experimental crosses, hybrid offspring from crosses in which S. multiplicata was maternal had significantly lower survival and longer development time than pure S. multiplicata offspring. By contrast, hybrid offspring from crosses in which S. bombifrons was maternal outperformed pure S. bombifrons offspring by reaching metamorphosis faster. These data suggest that, although S. multiplicata females are under selection to avoid hybridization, selection might favor those S. bombifrons females that hybridize with S. multiplicata if their breeding pond is highly ephemeral. Generally, the strength of selection to avoid hybridization may differ for hybridizing species, possibly impeding reinforcement.