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PubMedDate not supplied

Glyconeogenesis and urea synthesis in the toad Bufo viridis during acclimation to water restriction.

Metabolic consequences of osmotic stress were investigated in the toad Bufo viridis. Toads were acclimated either to terrestrial conditions in the absence of free water or to being partially immersed in 250 mmol L-1 NaCl, which was achieved by gradually increasing the salinity of the bath. This slow acclimation evoked little metabolic response, whereas the immediate osmotic challenge of water restriction resulted in a significant increase in the concentration of urea in the plasma and in liver glycogen. Urea accumulation, involving a transient increase in its rate of synthesis, allows the toads to lower their body water potential and thereby to absorb soil-bound water. The metabolic cost of this response is reduced by conserving the resulting by-product, glucose, as glycogen stored in the liver for future use.

Acclimatization↗
PubMed2002

Phosphoglucose isomerases of hagfish, zebrafish, gray mullet, toad, and snake, with reference to the evolution of the genes in vertebrates.

Phosphoglucose isomerase (PGI) is a protein with multiple functions. To infer its structure changes and evolution in vertebrates, we cloned cDNAs encoding PGI genes from hagfish (Paramyxine yangi), gray mullet (Mugil cephalus), zebrafish (Danio rerio), toad (Bufo melanosticus), and snake (Boiga kraepelini). Only one PGI gene was cloned in each of hagfish, toad, and snake, but two PGI genes were found in zebrafish and gray mullet, respectively. The PGI of hagfish encodes 554 amino acids, in contrast to the PGIs of bonyfishes, toad, and snake which encode 553 amino acids and the PGIs of mammals which encode 558 amino acids. Among 558 aligned amino acid sites, there are 314 sites (56.27%) totally conserved. To see if diversifying selection acts on PGI amino acids of vertebrates, we calculated the pairwise ratio of nonsynonymous versus synonymous substitution per site (Ka/Ks) and the ratio of radical amino acid changes versus conservative amino acid changes per sites (dR/dC) between PGI sequences. The average pairwise ratio between nonsynonymous substitutions per nucleotide (Ka) and synonymous substitutions per nucleotide (Ks) among vertebrate PGI sequences equals 0.047 +/- 0.019. The average pairwise ratio between radical amino acid changes and conservative amino acid changes (dR/dC) among the vertebrate PGIs equal 0.938 +/- 0.158 for charge changes, 0.558 +/- 0.085 for polarity changes, and 0.465 +/- 0.0714 when both polarity and volume are considered. There is no amino acid within the vertebrate PGIs under diversifying selection as analyzed by the method of Yang et al. (2000b). The results suggest that the present vertebrate PGIs are at evolutionary stasis and are being subjected to intense purifying selection. The purifying selection is to maintain polarity and volume of the protein but not the charge groups of amino acids. Phylogenetic analysis reveals that vertebrate PGIs can be classified into three major groups: the mammalian, amphibian-reptilian, and teleostean PGIs. The gene tree suggests that the gene duplication event of PGI in bonyfishes occurred before diversification of Acanthopterygii but after the split of bonyfishes and tetrapods. The evolution of multiple functions of PGI is discussed.

Amino Acid Sequence↗
PubMed1984

Occurrence and experimental infection of toads (Bufo marinus and B. granulosus) with Mycobacterium chelonei subsp. abscessus.

In a survey of 234 Amazonian toads and frogs, six strains of Mycobacterium chelonei subsp. abscessus were isolated from the liver or spleen of four of 66 Bufo marinus (6.1%) and from the kidney or peritoneal fluid of two of 86 B. granulosus (2.3%). There were no histopathological lesions in the viscera of the infected animals. Experimental infection of 29 captive B. marinus and B. granulosus, by the intraperitoneal route, with a pooled inoculum of M. chelonei subsp. abscessus caused five deaths near the end of a 2-month observation period. M. chelonei subsp. abscessus was isolated from the liver, spleen, kidney, gonad, heart and lung of toads killed at various intervals after inoculation, and intracellular acid-fast bacilli were seen in these organs. Histological evidence of invasion of tissues by mycobacteria became apparent from the 45th day after infection. The susceptibility to infection of B. marinus and B. granulosus suggests that these toads may serve as a fortuitous animal host for M. chelonei subsp. abscessus.

Animals↗
PubMed1990

Heat capacity and entropy changes of the major isotype of the toad (Bufo) parvalbumin induced by calcium binding.

The possible structural changes in the major isotype of parvalbumin from the toad (Bufo bufo japonicus) skeletal muscle caused by Ca2+ and Mg2+ binding have been analyzed by microcalorimetric titrations. Parvalbumin was titrated with Ca2+ in both the absence and presence of Mg2+ and with Mg2+ in the absence of Ca2+, at pH 7.0, and at 5 degrees, 15 degrees, and 25 degrees C. The two sites in a molecule were equivalent on Mg2(+)-Ca2+ exchange, but distinguishable on Ca2+ and Mg2+ binding. The reactions of parvalbumin with Ca2+ are exothermic at every temperature in both the absence and presence of Mg2+, but those with Mg2+ are always endothermic except for the binding to site 1 at 25 degrees C. The magnitudes of the hydrophobic and internal vibrational contributions to the heat capacity and entropy changes of parvalbumin on Ca2+ and Mg2+ binding and Mg2(+)-Ca2+ exchange have been estimated by the empirical method of Sturtevant [Sturtevant, J. M. (1977) Proc. Natl Acad. Sci. USA 74, 2236-2240]. Although no major conformational changes were noted between Ca2(+)- and Mg2(+)-bound forms of toad parvalbumin, the conformational difference was larger in Ca2+ (or Mg2+) binding to site 1 than site 2. This may indicate that the metal-free form is much less stable than any form with Ca2+ (or Mg2+) bound at one site at least. On Mg2(+)-Ca2+ exchange, the vibrational as well as hydrophobic entropy is only slightly increased in a parallel manner. In contrast, on Ca2+ (or Mg2+) binding, the hydrophobic entropy increases but the vibrational entropy decreases; the former indicates the sequestering of nonpolar groups from the surface to the interior of a molecule, and the latter suggests that the overall structures are tightened on Ca2+ (or Mg2+) binding but loosened on Mg2(+)-Ca2+ exchange. Despite the clear distinctions in the thermodynamic features, the conformational changes of toad parvalbumin are essentially the same as those of the two isotypes of bullfrog parvalbumins on Ca2+ binding and Mg2(+)-Ca2+ exchange.

Animals↗
PubMed1997

Effect of saponin treatment on the sarcoplasmic reticulum of rat, cane toad and crustacean (yabby) skeletal muscle.

1. Mechanically skinned fibres from skeletal muscles of the rat, toad and yabby were used to investigate the effect of saponin treatment on sarcoplasmic reticulum (SR) Ca2+ loading properties. The SR was loaded submaximally under control conditions before and after treatment with saponin and SR Ca2+ was released with caffeine. 2. Treatment with 10 micrograms ml-1 saponin greatly reduced the SR Ca2+ loading ability of skinned fibres from the extensor digitorum longus muscle of the rat with a rate constant of 0.24 min-1. Saponin concentrations up to 150 micrograms ml-1 and increased exposure time up to 30 min did not further reduce the SR Ca2+ loading ability of the SR, which indicates that the inhibitory action of 10-150 micrograms ml-1 saponin is not dose dependent. The effect of saponin was also not dependent on the state of polarization of the transverse-tubular system. 3. Treatment with saponin at concentrations up to 100 micrograms ml-1 for 30 min did not affect the Ca2+ loading ability of SR in skinned skeletal muscle fibres from the twitch portion of the toad iliofibularis muscle but SR Ca2+ loading ability decreased markedly with a time constant of 0.22 min-1 in the presence of 150 micrograms ml-1 saponin. 4. The saponin dependent increase in permeability could be reversed in both rat and toad fibres by short treatment with 6 microM Ruthenium Red, a potent SR Ca2+ channel blocker, suggesting that saponin does affect the SR Ca2+ channel properties in mammalian and anuran skeletal muscle. 5. Treatment of skinned fibres of long sarcomere length (> 6 microns) from the claw muscle of the yabby (a freshwater decapod crustacean) with 10 micrograms ml-1 saponin for 30 min abolished the ability of the SR to load Ca2+, indicating that saponin affects differently the SR from skeletal muscles of mammals, anurans and crustaceans. 6. It is concluded that at relatively low concentrations, saponin causes inhibition of the skeletal SR Ca2+ loading ability in a species dependent manner, probably by increasing the Ca2+ loss through SR Ca2+ release channels.

Animals↗
PubMed2000

Inhibition of basolateral cAMP permeability in the toad urinary bladder.

1. The effect of sulphonylurea drugs on hydrosmotic flow across toad urinary bladder epithelium was re-evaluated in the present study. Glibenclamide, added to the basolateral medium, significantly enhanced the osmotic flow induced by low doses of antidiuretic hormone (ADH) or forskolin (FK), while it inhibited the effect of exogenous cyclic adenosine monophosphate (cAMP) or its non-hydrolysable bromo derivative, 8-Br-cAMP, added to the basolateral medium. These opposite effects of glibenclamide on the transepithelial osmotic flow can be explained by a reduction of cAMP permeability across the basolateral membrane of the epithelium. The decrease in cAMP permeability leads, according to the direction of the cAMP gradient, to firstly an enhanced osmotic flow when cAMP is generated intracellularly by addition of ADH and FK, glibenclamide reducing cAMP exit from the cell, and secondly a decreased osmotic flow in response to cAMP (and 8-Br-cAMP) added to the basolateral medium, glibenclamide inhibiting, in this case, their entry into the cell. 2. The demonstration that glibenclamide actually inhibits the basolateral cAMP permeability rests on the fact that firstly it decreases the release of cAMP into the basolateral medium by about 40 %, at each concentration of ADH or forskolin tested, secondly it increases the cAMP content of paired hemibladders incubated in the presence of ADH or FK, when intracellular degradation was prevented by phosphodiesterase inhibition, and thirdly it decreases also the uptake of basolateral 8-Br-[3H]cAMP into paired toad hemibladders. 3. Taken together, the present data demonstrate that glibenclamide inhibits the toad urinary bladder basolateral membrane permeability to cAMP, most probably by a direct interaction with a membrane protein not yet indentified but distinct from the sulphonylurea receptor.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2004

A retrospective report of 90 dogs with suspected cane toad (Bufo marinus) toxicity.

OBJECTIVE: To report the clinical characteristics of toad toxicity in domestic dogs in Brisbane. DESIGN: A retrospective analysis of clinical cases. PROCEDURE: All cases of toad poisoning which presented to a northern suburbs emergency clinic in Brisbane over a 30-month period beginning in April 1999 were reviewed. RESULTS: A total of 90 canine cases of suspected toad poisoning were reviewed. Small breed dogs accounted for 76% of cases. Jack Russell, Silky, and Fox Terriers were the most represented breeds. Cases were reported year round, with fewest cases over the winter months. The most common clinical signs were increased salivation (78% of cases), and red oral mucous membranes (63% cases). Seizures occurred in 31% of cases. Generally the outcome was excellent with 96% survival.

Animals↗
PubMed1968

Amiloride: a potent inhibitor of sodium transport across the toad bladder.

1 Amiloride inhibits Na transport and short-circuit current (SCC) across the toad bladder. It is 1000 times more active at the mucosal than serosal surface. The lowest effective concentration was 10(-7)M.2. The inhibition was non-competitive with the sodium on the mucosal side of the bladder.3. Vasopressin, cyclic adenosine monophosphate (AMP) and aldosterone increased Na transport and SCC across the bladder and these effects were inhibited by amiloride.4. The antagonism of amiloride for vasopressin was non-competitive.5. Amphotericin B also increases Na transport across the bladder but its action was not changed by amiloride.6. Amiloride was without effects on SCC and diffusion potentials in bladders metabolically inhibited with CN(-) and iodoacetic acid (IAA).7. Neither plasma albumin, Ca(2+) nor adenosine triphosphate (ATP) altered the effects of amiloride.8. The only structural analogue of amiloride found to reduce SCC similarly was guanidine which was 1000 times less active. Pyrazine and a substituted pyrazine analogue were without effect. Neither guanidine nor the substituted pyrazine compound were competitive with amiloride.9. Amiloride had no effect on the osmotic permeability of the toad bladder either in the presence or absence of vasopressin.10. Na transport across the toad colon was also reduced by 10(-5)M amiloride at the mucosal surface.11. The possible mechanism of action of amiloride is discussed.

Adenine Nucleotides↗
PubMed1979

Light adaptation in toad rods: requirement for an internal messenger which is not calcium.

1. The mechanism of light adaptation was investigated by recording intracellularly from single rods in the isolated, superfused retina of the toad, Bufo marinus. Steady background lights produce decreases in rod sensitivity and changes in response wave form similar to those previously observed in the toad eyecup. 2. The sensitivity of a dark-adapted rod is halved by a background light which bleaches about 4 rhodopsins per rod per second. Since a toad rod contains over 2000 disks, a rhodopsin bleached in one disk must alter the effectiveness of rhodopsins bleached in others. This could occur if the state of adaptation in the rod were regulated by the concentration of some diffusable substance. 3. This diffusable substance cannot be Ca2+. Increases in intracellular Ca2+, produced experimentally either by increasing extracellular Ca2+ or by facilitating Ca2+ permeability into the rod with the ionophore X537A, cause a hyperpolarization of membrane potential and a decrease in response amplitude; but they do not produce changes in sensitivity and response wave form like those produced by background light. 4. Either Ca2+ is not the internal transmitter released from the disks during excitation, or the disks release or otherwise alter the concentration of a second diffusable substance, in addition to Ca2+, which regulates the state of adaptation.

Adaptation, Ocular↗
PubMed1983

Adapting lights and lowered extracellular free calcium desensitize toad photoreceptors by differing mechanisms.

Extracellular recordings were made across the outer segment layer of isolated, superfused toad retinas. Under these recording conditions, the photovoltage reflects primarily the current flowing through the outer-segment membrane of red rods. In normal toad Ringer solution, a dim conditioning flash desensitized a test flash response. The desensitization reached a peak 1.8-2.0 s after the conditioning flash and then declined approximately as an exponential with time constant 6 s. Lowered extracellular calcium, [Ca2+]o, desensitized the photoresponse. It required approximately ten times more light to reach a half-maximal response for each ten-fold change in [Ca2+]o from 10(-6) to 10(-9) M. When [Ca2+]o was less than 10(-7) M, substitution of Li+ for Na+ as the predominant monovalent cation in the superfusate permitted responses to continue and a resensitization of up to approximately 1 log unit was observed. The effects of lowered [Ca2+]o on response kinetics were markedly different from the effects of background lights producing a comparable desensitization. Low [Ca2+]o increased absolute latency and time-to-peak of the flash response. Background lights decreased time-to-peak, leaving latency unchanged. The effects of background lights and lowered [Ca2+]o are not additive. Moderate backgrounds had little effect on the intensity/response function in low [Ca2+]o. Conditioning flashes facilitated the test flash response in 10(-7) M-[Ca2+]o superfusate. These results can be understood in terms of the Ca2+ hypothesis of transduction (Hagins & Yoshikami, 1974) if it is assumed that lowered [Ca2+]o exposes an endogenous Ca2+ buffer. The data also provide evidence for a role of Na+/Ca2+ exchange in regulating intracellular Ca2+ concentration in the toad photoreceptor. A quantitative model based on these assumptions is derived and compared with the experimental data.

Action Potentials↗
PubMed1987

Response characteristics of pulmocutaneous arterial baroreceptors in the toad, Bufo marinus.

1. Response characteristics of baroreceptors with receptive fields in the pulmocutaneous artery (p.c.a.) were determined in pithed toads by applying pressure steps, ramps, sine waves, and volume infusions into the vascularly isolated and perfused p.c.a. 2. The baroreceptors exhibited phasic and tonic discharge thresholds (30.3 +/- 2.3 and 36.2 +/- 2.8 mmHg respectively) which were above mean arterial pressure values reported for conscious undisturbed toads. They are comparable, in this respect, to non-myelinated mammalian arterial baroreceptors. 3. The maximum-phasic and minimum-adapted discharge frequencies of the p.c.a. baroreceptors were low (30 and 2-3 spikes s-1, respectively), but resembled those reported for mammalian non-myelinated baroreceptors when an adjustment was made for the difference in the temperature of mammalian and amphibian preparations. 4. The sensitivity of the baroreceptor discharge frequency to pressure (delta F/delta P) was estimated from pressure-step and pressure-ramp stimuli. Both estimates were greater than those reported for mammalian systemic arterial baroreceptors after the values were normalized to the maximum discharge frequency of the receptors. delta F/delta P and saturation discharge frequency values estimated from ramp stimuli increased with the dP/dt of pressure ramps. 5. The diameter of the p.c.a. was measured by sonomicrometry in toads anaesthetized with urethane. The diameter pulsation was 7.7 +/- 0.3% of the mean diameter (3.5 +/- 0.1 mm) at a mean pulse pressure of 22 +/- 1 mmHg, and Peterson's pressure-strain modulus was calculated to be 4.0 X 10(5) +/- 0.3 X 10(5) dyn cm-2, which suggests that the p.c.a. is highly compliant, and in this respect is comparable to the pulmonary artery, but not to systemic arteries, in mammals. Baroreceptor discharge began near the point of peak dynamic compliance (dV/dP) and continued as dV/dP decreased. Increasing the rate of infusion reduced the peak dV/dP, but increased the baroreceptor discharge frequency. 6. The response to sinusoidal oscillating pressure stimuli was distorted by rectification. Increasing the frequency of sinusoidal stimulation over the range 0.05-1.0 cycles s-1 reduced the number of spikes per cycle, but increased the mean discharge frequency.

Action Potentials↗
PubMed1997

Relationship between depolarization-induced force responses and Ca2+ content in skeletal muscle fibres of rat and toad.

1. The relationship between the total Ca2+ content of a muscle fibre and the magnitude of the force response to depolarization was examined in mechanically skinned fibres from the iliofibularis muscle of the toad and the extensor digitorum longus muscle of the rat. The response to depolarization in each skinned fibre was assessed either at the endogenous level of Ca2+ content or after depleting the fibre of Ca2+ to some degree. Ca2+ content was determined by a fibre lysing technique. 2. In both muscle types, the total Ca2+ content could be reduced from the endogenous level of approximately 1.3 mmol l-1 (expressed relative to intact fibre volume) to approximately 0.25 mmol l-1 by either depolarization or caffeine application in the presence of Ca2+ chelators, showing that the great majority of the Ca2+ was stored in the sarcoplasmic reticulum (SR). Chelation of Ca2+ in the transverse tubular (T-) system, either by exposure of fibres to EGTA before skinning or by permeabilizing the T-system with saponin after skinning, reduced the lower limit of Ca2+ content to < or = 0.12 mmol l-1, indicating that 10-20% of the total fibre Ca2+ resided in the T-system. 3. In toad fibres, both the peak and the area (i.e. time integral) of the force response to depolarization were reduced by any reduction in SR Ca2+ content, with both decreasing to zero in an approximately linear manner as the SR Ca2+ content was reduced to < 15% of the endogenous level. In rat fibres, the peak size of the force response was less affected by small decreases in SR content, but both the peak and area of the response decreased to zero with greater depletion. In partially depleted toad fibres, inhibition of SR Ca2+ uptake potentiated the force response to depolarization almost 2-fold. 4. The results show that in this skinned fibre preparation: (a) T-system depolarization and caffeine application can each virtually fully deplete the SR of Ca2+, irrespective of any putative inhibitory effect of SR depletion on channel activation; (b) all of the endogenous level of SR Ca2+ must be released in order to produce a maximal response to depolarization; and (c) a substantial part (approximately 40%) of the Ca2+ released by a depolarization is normally taken back into the SR before it can contribute to force production.

Animals↗
PubMed1960

Perception of apparent motion in the common toad.

A simple and inexpensive apparatus makes possible the feeding of nonliving objects to the toad. The device is used to demonstrate the perception of apparent or "induced" motion. Two methods are successful: (i) toad and food moving together at a constatnt velocity in a stationary environment; (ii) toad and food stationary with the environment moving. The phenomena are similar to those found in human beings.

Animals↗
PubMed1983

Actin filaments and vasopressin-stimulated water flow in toad urinary bladder.

Vasopressin increases the water permeability of the apical membrane of the granular epithelial cells of the toad urinary bladder. Cytochalasin B inhibits this action of the hormone, indicating that microfilaments may play a role in the water permeability response. We have extended previous functional studies with cytochalasin B and have demonstrated that dihydrocytochalasin B, a more specific inhibitor of actin filament elongation, similarly diminishes the hydrosmotic response to vasopressin. Biochemical studies of isolated epithelial cells indicate that an actin-like protein accounts for about 10% of the soluble protein of the epithelium. Morphological studies of whole toad bladders incubated with heavy meromyosin conclusively demonstrate that actin is a component of the epithelial cells and that actin-containing filaments are associated with both plasma membranes and cytoplasmic organelle membranes. Taken together, these findings provide strong, albeit indirect, evidence that actin microfilaments play a functional role in the hormone-induced increase in water permeability in the toad urinary bladder.

Actins↗
PubMed1988

Purification of PRL receptors from toad kidney: comparisons with rabbit mammary PRL receptors.

The binding characteristics of the prolactin (PRL) receptors present in toad (Bufo marinus) kidneys were investigated and compared to those of PRL receptors present in rabbit mammary glands. The molecular characteristics of the Triton X-100 solubilized renal and mammary PRL receptors were assessed by gel filtration and by migration analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after affinity labeling of the binding sites with 125I-human growth hormone. Similar results were obtained for both receptors. Partial purification of the toad PRL receptor could be achieved by affinity chromatography. The molecular weight of this purified receptor could be determined by analysis on SDS-PAGE. With the use of a polyclonal antiserum raised against a purified preparation of rabbit mammary PRL receptor, one or several antigenic epitope(s) could be identified on the core of the toad renal PRL receptor. In conclusion, although the structure and the biological role(s) of PRL have substantially changed during evolution, the receptor for this hormone has retained many of its structural features as could be assessed between an amphibian and a mammalian species on functionally different target tissues.

Affinity Labels↗
PubMed1991

Oxygen uptake of isolated toad skin epithelium: micromeasurement and effect of ionic acclimation.

Oxidative metabolism of isolated toad skin epithelium (Bufo viridis) was investigated in vitro under open-circuit conditions using the spectrophotometric oxyhemoglobin micromethod. This highly sensitive technique has been adapted for studying several epithelia in parallel and for detecting possible regional variations of oxygen uptake in individual epithelium. Changes in the proportion of mitochondria-rich cells (MRC) by ionic acclimation affected oxidative metabolism under nontransporting condition. After acclimation of animals to either NaNO3 or NaCl solutions (100 mmol/l, for greater than 2 wk), the number of MRC per square millimeter in epithelia from nonacclimated and NaNO3- and NaCl-acclimated animals was 350 +/- 113, 460 +/- 196, and 107 +/- 52, respectively. O2 uptake of nonacclimated and NaNO3-acclimated epithelia was significantly higher than that of NaCl-acclimated epithelia (i.e., 0.89 and 0.90 vs. 0.57 nmol O2.h-1.mm-2, respectively). The correlation established between O2 uptake and number of MRC allowed evaluation of the respiration rate of one single MRC, i.e., approximately 1 pmol O2/h. The lowest mitochondrial oxidative activity was found in the epithelia from NaCl-acclimated toads where the uncoupler 2,4-dinitrophenol (50 mumols/l) had the highest relative stimulatory effect (+114%). Acetazolamide (50 mumols/l), a potent inhibitor of carbonic anhydrase mainly present in the MRC, reduced selectively by 31% O2 uptake of the MRC-rich epithelia (NaNO3 acclimated). O2 uptake increased significantly by approximately 80% when basolateral pH increased from 5.8 to 7.8, but did not depend on apical pH. These findings indicate that under nontransporting (open-circuit) conditions, aerobic metabolism of the isolated toad skin epithelium is related to the density and/or characteristics of the MRC.(ABSTRACT TRUNCATED AT 250 WORDS)

Acclimatization↗
PubMed1992

Water and nonelectrolyte permeabilities of apical membranes of toad urinary bladder granular cells.

Certain types of epithelial cells such as those lining the toad urinary bladder have been classified as "tight" because their apical membranes exhibit low permeabilities to water, ions, and small nonelectrolytes. However, the permeability properties and structural features of these specialized apical membranes remain unclear because these membranes have never been purified. To isolate toad bladder granular cell apical membranes, we derivatized the bladder apical surface with the membrane-impermeant bifunctional reagent N-hydroxysulfosuccinimydyl-S,S-biotin (NHS-SS-biotin). After cell disruption, these derivatized apical membranes were purified using streptavidin-coated magnetic beads in a magnetic field. With the use of lactoperoxidase-mediated radioiodination as a marker for apical membrane, this preparative procedure purified apical membrane 48- or 72-fold as compared with homogenate. Thin section electron microscopy revealed unilamellar vesicles with some nonvesiculated membranes, while fragments of organelles such as mitochondria were absent. Water and nonelectrolyte permeabilities of purified apical membrane vesicles were similar to those obtained in intact bladders in the absence of antidiuretic hormone stimulation. The results demonstrate that isolated apical vesicles do not contain water channels and confirm the applicability of Overton's rule to the apical membrane of the toad urinary bladder. The technique has general applicability to isolation of other plasma membranes, and the apical membranes obtained are suitable for structural analysis.

Animals↗
PubMed2000

Excitation, inhibition, and suppression by odors in isolated toad and rat olfactory receptor neurons.

Vertebrate olfactory receptor neurons (ORNs) exhibit odor-induced increases in action potential firing rate due to an excitatory cAMP-dependent current. Fish and amphibian ORNs also give inhibitory odor responses, manifested as decreases in firing rate, but the underlying mechanism is poorly understood. In the toad, an odor-induced Ca(2+)-activated K(+) current is responsible for the hyperpolarizing receptor potential that causes inhibition. In isolated ORNs, a third manner by which odors affect firing is suppression, a direct and nonspecific reduction of voltage-gated and transduction conductances. Here we show that in whole cell voltage-clamped toad ORNs, excitatory or inhibitory currents were not strictly associated to a particular odorant mixture. Occasionally, both odor effects, in addition to suppression, were concurrently observed in a cell. We report that rat ORNs also exhibit odor-induced inhibitory currents, due to the activation of a K(+) conductance closely resembling that in the toad, suggesting that this conductance is widely distributed among vertebrates. We propose that ORNs operate as complex integrator units in the olfactory epithelium, where the first events in the process of odor discrimination take place.

Animals↗