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PubMed1979

Excretion of phenol red by the Necturus kidney.

Phenol red (phenolsulfonphthalein, PSP) is thought to be secreted by proximal kidney tubules in all vertebrates. The present study examined PSP transport by the kidney of the salamander, Necturus maculosus. In Necturus kidneys perfused with oxygenated Ringer solution, the PSP/creatinine clearance ratio was unity. Perfusion with 1 mM octanoate converted net p-aminohippurate (PAH) reabsorption to net secretion, but had no effect on PSP. In seven urethan-anesthetized Necturi, the PSP/inulin clearance ratio averaged 0.85 +/- 0.21 (SD), not significantly different from unity. Thin slices from Necturus kidneys incubated in vitro for 2 h failed to accumulate PSP; slice-to-medium (S/M) concentration ratios averaged 0.8 +/- 0.2 (n = 6). With frog kidney slices, (S/M)PSP was 9.6 +/- 1.4 (n = 6). Necturus kidney slices accumulated PAH ((S/M)PAH = 4.1 +/- 0.7) (n = 6), but uptake was not inhibited by 1 mM PSP. We conclude that Necturus kidney tubules transport PAH, but do not transport PSP. These results are consistent with the hypothesis that the organic acid secretory system in most animals involves several carriers.

Aminohippuric Acids↗
PubMed1991

Ca(2+)-dependent chloride conductance in Necturus taste cells.

This report describes the occurrence and localization of a Ca(2+)-dependent chloride conductance in taste cells of Necturus maculosus. Lingual epithelium from Necturus was removed with blunt dissection and mounted in a modified Ussing chamber which allowed individual taste cells to be impaled with intracellular micropipettes. Solutions in the mucosal and serosal chambers could be changed independently and the properties of apical and basolateral membranes tested separately. Action potentials in taste cells, elicited by brief depolarizing current pulses passed through the intracellular recording microelectrode, provided an accurate description of whether voltage-dependent conductances had been blocked or unmasked by the experimental conditions. We found that Ca2+ influx during the action potential triggers a prolonged depolarization due to Ca(2+)-dependent conductance changes, particularly in the presence of TEA to block repolarizing K+ currents. This afterdepolarization could last up to 7 sec and is due, in part, to a Ca(2+)-dependent Cl- conductance. Other Ca(2+)-dependent channels such as Ca(2+)-dependent K+ channels or nonselective cation channels may also contribute to the afterpotential. Calcium-dependent conductance channels were situated on apical and basolateral membranes of the taste cells. We speculate that Ca(2+)-dependent Cl- channels may play a role in discriminating chloride salts from salts of other anions and may help shape receptor cell responses elicited by taste stimuli.

Action Potentials↗
PubMed1999

Morphological variation of hypaxial musculature in salamanders (Lissamphibia: caudata).

Despite the acknowledged importance of the locomotory and respiratory functions associated with hypaxial musculature in salamanders, variation in gross morphology of this musculature has not been documented or evaluated within a phylogenetic or ecological context. In this study, we characterize and quantify the morphological variation of lateral hypaxial muscles using phylogenetically and ecologically diverse salamander species from eight families: Ambystomatidae (Ambystoma tigrinum), Amphiumidae (Amphiuma tridactylum), Cryptobranchidae (Cryptobranchus alleganiensis), Dicamptodontidae (Dicamptodon sp.), Plethodontidae (Gyrinophilus porphyriticus), Proteidae (Necturus maculosus), Salamandridae (Pachytriton sp.), and Sirenidae (Siren lacertina). For the lateral hypaxial musculature, we document 1) the presence or absence of muscle layers, 2) the muscle fiber angles of layers at mid-trunk, and 3) the relative dorsoventral positions and cross-sectional areas of muscle layers. Combinations of two, three, or four layers are observed. However, all species retain at least two layers with opposing fiber angles. The number of layers and the presence or absence of layers vary within species (Necturus maculosus and Siren lacertina), within genera (e.g., Triturus), and within families. No phylogenetic pattern in the number of layers can be detected with a family-level phylogeny. Fiber angle variation of hypaxial muscles is considerable: fiber angles of the M. obliquus externus range from 20-80 degrees; M. obliquus internus, 14-34 degrees; M. transversus abdominis, 58-80 degrees (acute angles measured relative to the horizontal septum). Hypaxial musculature comprises 17-37% of total trunk cross-sectional area. Aquatic salamanders show relatively larger total cross-sectional hypaxial area than salamanders that are primarily terrestrial.

Anatomy, Cross-Sectional↗
PubMed1990

Radial density distribution of chromatin: evidence that chromatin fibers have solid centers.

Fiber diameter, radial distribution of density, and radius of gyration were determined from scanning transmission electron microscopy (STEM) of unstained, frozen-dried chromatin fibers. Chromatin fibers isolated under physiological conditions (ionic strength, 124 mM) from Thyone briareus sperm (DNA linker length, n = 87 bp) and Necturus maculosus erythrocytes (n = 48 bp) were analyzed by objective image-processing techniques. The mean outer diameters were determined to be 38.0 nm (SD = 3.7 nm; SEM = 0.36 nm) and 31.2 nm (SD = 3.6 nm; SEM = 0.32 nm) for Thyone and Necturus, respectively. These data are inconsistent with the twisted-ribbon and solenoid models, which predict constant diameters of approximately 30 nm, independent of DNA linker length. Calculated radial density distributions of chromatin exhibited relatively uniform density with no central hole, although the 4-nm hole in tobacco mosaic virus (TMV) from the same micrographs was visualized clearly. The existence of density at the center of chromatin fibers is in strong disagreement with the hollow-solenoid and hollow-twisted-ribbon models, which predict central holes of 16 and 9 nm for chromatin of 38 and 31 nm diameter, respectively. The cross-sectional radii of gyration were calculated from the radial density distributions and found to be 13.6 nm for Thyone and 11.1 nm for Necturus, in good agreement with x-ray and neutron scattering. The STEM data do not support the solenoid or twisted-ribbon models for chromatin fiber structure. They do, however, support the double-helical crossed-linker models, which exhibit a strong dependence of fiber diameter upon DNA linker length and have linker DNA at the center.

Animals↗
PubMed1990

The diameters of frozen-hydrated chromatin fibers increase with DNA linker length: evidence in support of variable diameter models for chromatin.

The diameters of chromatin fibers from Thyone briareus (sea cucumber) sperm (DNA linker length, n = 87 bp) and Necturus maculosus (mudpuppy) erythrocytes (n = 48 bp) were investigated. Soluble fibers were frozen into vitrified aqueous solutions of physiological ionic strength (124 mM), imaged by cryo-EM, and measured interactively using quantitative computer image-processing techniques. Frozen-hydrated Thyone and Necturus fibers had significantly different mean diameters of 43.5 nm (SD = 4.2 nm; SEM = 0.61 nm) and 32.0 nm (SD = 3.0 nm; SEM = 0.36 nm), respectively. Evaluation of previously published EM data shows that the diameters of chromatin from a large number of sources are proportional to linker length. In addition, the inherent variability in fiber diameter suggests a relationship between fiber structure and the heterogeneity of linker length. The cryo-EM data were in quantitative agreement with space-filling double-helical crossed-linker models of Thyone and Necturus chromatin. The data, however, do not support solenoid or twisted-ribbon models for chromatin that specify a constant 30 nm diameter. To reconcile the concept of solenoidal packing with the data, we propose a variable-diameter solid-solenoid model with a fiber diameter that increases with linker length. In principle, each of the variable diameter models for chromatin can be reconciled with local variations in linker length.

Animals↗
PubMed1989

A specific androgen-binding protein (ABP) in Necturus testis and its zonal distribution.

The urodele amphibian Necturus maculosus has a zoned testis, which is advantageous for separating Leydig cells from germinal elements and for studying stage-dependent biochemical changes. Using [3H]testosterone (T) in a standard binding assay and dextran-coated charcoal (DCC) or Sephadex LH-20 to separate free and bound steroids, we identified an androgen-binding protein (ABP) in Necturus testis cytosols. This protein was of high affinity (Kd = 10(-9) M) and was saturable (Bmax = 10(-9) M) and specific for androgen (T; 5 alpha-dihydrotestosterone, DHT) but could be distinguished from the androgen receptor of Necturus testis by its relative abundance (300-550 fmol/mg protein), short half-time of dissociation (3 min at 22 degrees C), inability to adhere to DNA-cellulose, and absence from nuclear extracts. Additionally, when analyzed on sucrose gradients, the ABP of Necturus testis sedimented at 6-7 S in both low or high ionic strength buffers. In that estradiol (E2) is a poor competitor for T-binding, this protein resembles a sex steroid-binding protein previously identified in urodele serum but differs from the ABP and testosterone-estradiol-binding globulin (TEBG) of rodents, humans, goldfish, and sharks. It is differentially distributed within the testis, with the highest levels in immature lobular regions composed of Sertoli cells and germ cells in premeiotic stages and lower levels in regions composed primarily of Leydig cells. The cellular source and function of this protein in Necturus testis remain to be determined.

Androgen-Binding Protein↗
PubMed1991

Immunocytochemistry of gamma-aminobutyric acid, glutamate, serotonin, and histamine in Necturus taste buds.

Little information is currently available about which neurotransmitters are involved in signal processing in the peripheral sensory organs of taste, taste buds. Synaptic contacts between taste cells and sensory axons have long been known to exist, but what substances are active at these synapses is not known. Our objective in this study was to test for the presence of the neurotransmitter candidates, GABA, glutamate, serotonin, and histamine in taste buds of Necturus maculosus. Light microscopic immunocytochemical techniques were used to investigate the location of these substances in taste buds and surrounding epithelium. GABA and glutamate were detected in nerve fibers that innervate the taste buds, and, to a substantially lesser extent, in fine, varicose axons that penetrated the surrounding nontaste epithelium. Serotonin immunostaining was strong in basal cells in frog taste discs but was only faintly detected in Necturus taste buds. Histamine was not detected at all in taste buds. We conclude that amino acid neurotransmission may be involved in taste mechanisms and that monoamines may also play a role in chemosensory transduction in the taste bud. On the basis of our inability to detect histamine with immunocytochemical techniques, we conclude that this substance is unlikely to be a major neurotransmitter in Necturus taste buds.

Animals↗
PubMed1983

Regenerative impulses in taste cells.

Taste cells and nongustatory epithelial cells in the isolated lingual mucosa from the mud puppy Necturus maculosus were impaled with microelectrodes. The taste cells, but not surrounding epithelial cells, were electrically excitable when directly stimulated with current passed through the recording electrode. Action potentials produced by taste cells had both a sodium and a calcium component.

Action Potentials↗
PubMed1999

Isolated epithelial cells from amphibian urinary bladder express functional gap junctional hemichannels.

Exposure of the urinary bladder epithelium of Necturus maculosus (NUB) to protease and collagenase yields approximately 50% isolated polarized cells. These cells express a membrane current slowly activated by depolarization or by removal of external divalent cations. The biophysical and pharmacological properties of the current are largely consistent with those of gap junctional hemichannels. After removal of divalent cations, the cells can also be loaded with 5(6)-carboxyfluorescein, a hydrophilic fluorescent anionic dye, and exposure to dye reduces the current in a manner dependent on membrane voltage and side of application. In contrast, Necturus gallbladder (NGB) cells exhibit no membrane conductance attributable to gap junctional hemichannels, although previous studies reveal the persistence of gap junction plaques on the plasma membrane. We conclude that functional gap junctional hemichannels can be expressed on the surface of certain isolated epithelial cells and that this is not a necessary consequence of the isolation procedure. These structures may contribute to cell damage under pathological conditions involving cell detachment.

Animals↗
PubMed2000

Potassium channels in basolateral membrane vesicles from necturus enterocytes: stretch and ATP sensitivity.

We have previously reported that ATP-inhibitable K(+) channels, in vesicles derived from the basolateral membrane of Necturus maculosus small intestinal cells, exhibit volume regulatory responses that resemble those found in the intact tissue after exposure to anisotonic solutions. We now report that increases in K(+) channel activity can also be elicited by exposure of these vesicles to isotonic solutions containing glucose or alanine that equilibrate across these membranes. We also demonstrate that swelling after exposure to a hypotonic solution or an isotonic solution containing alanine or glucose reduces inhibition of channel activity by ATP and that this finding cannot be simply attributed to dilution of intravesicular ATP. We conclude that ATP-sensitive, stretch-activated K(+) channels may be responsible for the well-established increase in basolateral membrane K(+) conductance of Necturus small intestinal cells after the addition of sugars or amino acids to the solution perfusing the mucosal surface, and we propose that increases in cell volume, resulting in membrane stretch, decreases the sensitivity of these channels to ATP.

Adenosine Triphosphate↗
PubMed1986

Catecholamine, serotonin, and substance P-like peptide containing intrinsic neurons in the mudpuppy parasympathetic cardiac ganglion.

The mudpuppy cardiac ganglion contains 2 neuron types: large parasympathetic postganglionic projection neurons and smaller intrinsic neurons originally described by McMahan and Purves (1976) as intensely fluorescent (SIF) cells. The function of these SIF cells, present in the mudpuppy cardiac ganglion, is unknown. Further, direct application of catecholamines, which are thought to be contained in SIF cells, to the parasympathetic postganglionic cells has no effect (Hartzell et al., 1977). As SIF cells in other ganglion preparations recently have been shown to contain putative transmitter substances in addition to catecholamines, immunocytochemical experiments were conducted to test for the presence of additional transmitter substances in the SIF cells within the cardiac ganglion. Whole-mount septal preparations were dissected from Necturus maculosus and processed for indirect immunocytochemistry. The results indicated that many of these intrinsic neurons contained 5-HT or a substance P-like peptide, or both. Many small intrinsic neurons which contain either substance P or 5-HT were also positive for aqueous-aldehyde-induced fluorescence, indicating the presence of a catecholamine. Finally, some of these cells appeared to contain all 3: a catecholamine, 5-HT, and a substance P-like peptide.

Animals↗
PubMed1977

Effects of radiation upon the light-sensing elements of the retina as characterized by scanning electron microscopy.

A model system using Necturus maculosus, the common mudpuppy, was established for evaluating effects of radiation upon the light-sensing elements of the retina. Accelerated heavy ions of helium and neon from the Berkeley Bevalac were used. A number of criteria were chosen to characterize radiation damage by observing morphological changes with the scanning electron microscope. The studies indicated retina sensitivity to high-LET (neon) particles at radiation levels below 10 rads (7 particles per visual element) whereas no significant effects were seen from fast helium ions below 50 rads.

Animals↗
PubMed1993

Independently gated multiple substates of an epithelial chloride-channel protein.

We have purified a protein from Necturus maculosus gallbladder cells that forms chloride channels in an artificial membrane. The same protein apparently can form channels that are highly selective for chloride but can have conductances varying from 9 to about 150 pS. The high-conductance channels are blocked by the monoclonal antibody used to purify the protein, but this antibody has no effect on the 9-pS channels. The observation that gating of the low- and high-conductance states is independent and that the antibody affects only the latter has implications regarding the control of chloride conductance in cell membranes and the different types of channels described in those cells.

Animals↗