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PubMed1983

Influence of Ca2+ on the mechanosensitivity of the hair cells in the lateral line organs of Necturus maculosus.

The degree of synchronization (DOS) between the afferent spike activity from one stitch of the lateral line of Necturus maculosus (in vivo) and the mechanical stimulation of one neuromast of the same stitch was measured under different circumstances. The DOS was found to be independent of changes in the concentration of monovalent cations (Na+, K+ and choline+) in the bulk solution at high Ca concentration (1 mM). DOS was also independent of the Ca concentration in the range 1 mM-1 microM in Tris-HCl buffer, but was markedly reduced at Ca = 10 microM in MOPS-KOH buffer. The reduced DOS, however, could be restored by addition of 10-20 mM KCl. 5 mM of 4-aminopyridine did not influence the DOS at high Ca concentration, but completely reduced DOS at Ca = 10 microM. D600 (a methoxy derivative of verapamil) decreased DOS both at high and low Ca concentration.

4-Aminopyridine↗

PubMed2005

Analyzing the radiation of the melanocortins in amphibians: cloning of POMC cDNAs from the pituitary of the urodele amphibians, Amphiuma means and Necturus maculosus.

Proopiomelanocortin (POMC) cDNAs were cloned and sequenced from brain extracts of two species of urodele amphibians: Amphiuma means and Necturus maculosus. Although the two species of urodele amphibians belong to separate families, and do not share a direct common ancestor, the level of primary sequence identity for the open reading of the POMC cDNAs was 90% at the amino acid level and 79% at the nucleotide level. It appears that the POMC gene in these urodele amphibians has been accumulating mutations at the amino acid level at a slower rate than the POMC gene in other sarcopterygian orders.

Amino Acid Sequence↗

PubMed1984

Electron microscopic study of the innervation of the renal tubules and urinary bladder epithelium in Rana catesbeiana and Necturus maculosus.

The fine structure of the kidney and the bladder of the bullfrog (Rana catesbeiana), the bullfrog tadpole, and the mudpuppy (Necturus maculosus) were studied with special attention to the innervation of renal tubule cells and bladder epithelial cells. In the bullfrog kidney, nerve terminals and varicosities were frequently associated with the tubule cells, apparently in an increasing order from the proximal tubule to the connecting tubule. Although these terminals and varicosities did not directly contact the tubular cell membrane, an aggregation of synaptic vesicles on the side facing the tubule was considered as morphological evidence that neurotransmitter can be released here and can affect the transport activity of the tubule cells. The association of nerve varicosities with canaliculi cells in the connecting tubule was also demonstrated. In the bullfrog tadpoles, renal tubule cells were occasionally innervated. In the mudpuppy, renal tubule cells were only poorly innervated. The epithelium of the bullfrog bladder was commonly innervated. Nerve terminals with synaptic vesicles were located very near basal cells and even contacted them directly on rare occasions. In the mudpuppy, the innervation of the bladder epithelium was observed infrequently. The bullfrog tadpoles did not possess an apparent bladder. In all materials studied, renal arterioles and bladder smooth muscle cells were innervated.

Animals↗

PubMedDate not supplied

Isolation and primary culture of Necturus maculosus (Amphibia: Urodela) hepatocytes.

In order to evaluate their suitability for physiological and ecotoxicological studies, hepatocytes were isolated from the common mudpuppy (Necturus maculosus) using a two-step collagenase perfusion. Hepatocytes in primary culture were investigated for 14 d using light and electron microscopy and biochemical analyses. A typical perfusion yielded 1.7 x 10(5) viable hepatocytes per gram body weight with an average viability of 86 +/- 5%. The majority of isolated cells remained in suspension and formed aggregates. The viability of hepatocytes in primary culture was dependent on a fetal calf serum (FCS) concentration and incubation temperature. Viability was best at 8 degrees C in Leibovitz L-15 medium supplemented with 5% FCS. The ultrastructural characteristics of freshly isolated hepatocytes resembled those of N. maculosus hepatocytes in vivo. Whereas hepatocyte viability remained relatively stable (around 80%) up to 14 d in culture, electron microscopic analyses revealed changes at ultrastructural level. The majority of hepatocytes retained similar structural characteristics to those in vivo up to 4 d. Loss of cellular polarity, fractionation of rough endoplasmic reticulum, formation of autophagosomes, and successive exhaustion of cellular glycogen deposits were observed with increased time in culture. Functional integrity, as estimated by tyrosine aminotransferase induction, decreased during the culture period. Ultrastructural and biochemical analyses indicate the need for further improvement of culture conditions. Nevertheless, isolated hepatocytes in primary culture for up to 4 d can be recommended as a model for physiological and toxicological studies in lower vertebrates.

Animals↗

PubMed1994

Cotransport of K+, Cl- and H2O by membrane proteins from choroid plexus epithelium of Necturus maculosus.

1. The interaction between K+, Cl- and H2O fluxes was studied in the ventricular membrane of the choroid plexus epithelium from Necturus maculosus by means of ion-selective microelectrodes. 2. Three experimental strategies were adopted: the osmolarity of the ventricular solution was increased abruptly by addition of (i) mannitol or (ii) KCl; (iii) Na+ in the ventricular solution was replaced isosmotically by K+. 3. The mannitol experiments showed that H2O had two pathways across the ventricular membrane. One was purely passive, with a water permeability, L'p, of 0.64 x 10(-4) cm s-1 (osmol l-1)-1. This operated in parallel with an ion-dependent pathway of similar magnitude which was abolished in Cl(-)-free solutions. 4. When KCl was added there was a flow of H2O into the cell. Surprisingly, this took place despite the osmotic gradient which favoured an efflux of H2O. The effect was blocked by frusemide (furosemide), in which case KCl had the same effects as applications of NaCl or mannitol. 5. Replacement of Na+ with K+ caused an influx of H2O. This flux could proceed against osmotic gradients implemented by mannitol. 6. The present data and those of earlier publications show that the interdependence of the fluxes of K+, Cl- and H2O in the exit membrane can be described as cotransport. The fluxes have a fixed stoichiometry of 1:1:500, the flux of one species is able to energize the flux of the two others, and the transport exhibits saturation and is specific for K+ and Cl-. 7. A molecular model based upon a mobile barrier in a membrane spanning protein gives an accurate quantitative description of the data.

Animals↗

PubMed1991

Secondary active transport of water across ventricular cell membrane of choroid plexus epithelium of Necturus maculosus.

1. The interaction between Cl-, K+ and H2O fluxes were studied in the ventricular membrane of the choroid plexus epithelium from Necturus maculosus by means of ion-selective microelectrodes. The flux of H2O was measured by means of K+ electrodes as the dilution or concentration of intracellular choline ions, Ch+i. 2. In one series of experiments Cl- was readministered to the ventricular solution of tissues incubated in media with low Cl- concentrations. The resulting influx of Cl- was associated with an instantaneous influx of K+ and H2O. 3. Both the Cl- and the K+ influxes were reduced by the diuretic furosemide but were unaffected by inhibitors of Na+, K(+)-ATPase or changes in membrane potentials induced by Ba2+. Since the influx of K+ proceeds against its electrochemical gradient and is unaffected by changes in membrane potentials, the membrane exhibits secondary active, electroneutral transport of K+. 4. The influx of water, initiated simultaneously with the influx of K+ and Cl-, commenced before these ions had changed the osmolarity of the intracellular solution significantly. The influx of H2O could proceed against an osmotic gradient. The influx stopped when 100 mmol l-1 of mannitol was added to the ventricular solution at the same time as the Cl- ions. The influx of H2O was inhibited by K+ removal, furosemide or high external Ba2+ (10 mmol l-1), but not by strophanthidin, ouabain or low concentrations of Ba2+ (0.5 mmol l-1). The influx could not continue with other permeable anions, NO3-, acetate- or SCN-, replacing Cl-. 5. In another series of experiments Cl- was removed from the ventricular solution of tissues bathed in saline solutions with normal concentrations of Cl-. The resulting efflux of Cl- was associated with an instantaneous efflux of K+ and H2O. This efflux of H2O could proceed against an osmotic gradient of up to 70 mosmol l-1. This effect was inhibited by furosemide, in which case the water fluxes were entirely dependent on the osmotic gradients and the osmotic water permeability Lp of the ventricular membrane. 6. The data suggest that there is a coupling between the flux of KCl and of water in the ventricular membrane, which implies that the reflection coefficient sigma for KCl under the given circumstances is less than one. I suggest that the ability of leaky epithelia to transport against osmotic gradients depends on such a coupling, which derives from the properties of the proteins through which K+, Cl- and H2O leave the cell.

Animals↗

PubMedDate not supplied

Carbonic anhydrase and red blood cell anion exchange in the neotenic aquatic salamander, Necturus maculosus.

Carbonic anhydrase (CA) activity in blood and other tissues and red blood cell (rbc) anion exchange were measured in the mud puppy, Necturus maculosus, in order to gain insight into the strategy for CO2 transport used by these neotenic salamanders and to further explore evolutionary relationships between rbc CA activity and anion exchange in nonmammalian vertebrates. CA activity was detectable in all of the tissues examined, but CA activity in blood was much lower than that in most vertebrates. There was no indication, however, that additional CA had been incorporated into the membrane fraction of other tissues to compensate for this low blood CA activity. In further contrast to most other animals, low levels of CA activity were also detectable in mud puppy plasma. Preliminary characterization of the rbc CA indicated that the Type II, fast-turnover enzyme was indeed present, but that there are a very low number of active sites in mud puppy rbc's. Further experiments showed that the rbc's were highly permeable to anions and that the relative rate of anion flux could be inhibited by 4, 4-diisothiocyanostilbene-2,2-disulphonic acid. Thus, the process of CO2 transport in the blood of mud puppies probably involves components of the Jacobs-Stewart cycle, as in most other vertebrates.

Adaptation, Physiological↗

PubMed1975

Micropuncture study of the renal responses of the urodele amphibian Necturus maculosus to injections of arginine vasotocin and an anti-aldosterone compound.

1. Necturus maculosus kidney function has been examined using standard clearance techniques and renal tubular micropuncture methodology. 2. Throughout, cyanocobalamin (vitamin B12) has been used to monitor glomerular filtration rate (GFR) and tubular water movements. It was established that this substance was handled by the Necturus kidney in a similar manner to inulin. It can be readily analysed, together with renal electrolytes, by electron microprobe techniques. 3. Profiles of transtubular gradients (TF:P ratios) along the nephron were established for osmolarity, sodium, potassium, calcium and cobalt (of cyanocobalamin). 4. Ureteral urine is always hyposmotic with respect to plasma and the site of dilution of the plasma ultrafiltrate is within the distal segment. 5. Up to 30% of the filtrate is isosmotically reabsorbed along the proximal tubule; the tubular fluid:plasma ratio for osmolarity and sodium is around 1, and the TF:P for cobalt of cyanocobalamin is about 1.4 by the end of this segment. 6. The renal effects of the neurohypophysial hormone arginine vasotocin (AVT) and an aldosterone antagonist (SC14266; Soldactone) have been examined. 7. AVT was consistently antidiuretic causing both a decreased GFR and an enhanced distal tubular reabsorption of water. 8. SC14266 also increased distal tubular reabsorption of water. Such an effect differs from that found in higher vertebrates, and may indicate a "glucocorticoid-type" of renal action for aldosterone in amphibians.

Aldosterone↗

PubMed1992

Immunocytochemical survey of putative neurotransmitters in taste buds from Necturus maculosus.

To investigate synaptic mechanisms in taste buds and collect information about synaptic transmission in these sensory organs, we have examined taste buds of the mudpuppy, Necturus maculosus for the presence of neurotransmitters and neuromodulators. Immunocytochemical staining at the light microscopic level revealed the presence of serotonin-like and cholecystokinin-like (CCK) immunoreactivity in basal cells in the taste bud. Nerve fibers innervating taste buds were immunoreactive for vasoactive intestinal peptide-like (VIP), substance P-like, and calcitonin gene-related peptide-like (CGRP) or compounds closely related to these substances. Immunoreactivity for tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) in the taste cells and nerve fibers was absent. These data suggest that serotonin, CCK, VIP, substance P, and CGRP are involved in synaptic transmission or neuromodulation in the peripheral organs of taste. No evidence was found for cholinergic or adrenergic mechanisms on the basis of the absence of immunocytochemical staining for key enzymes involved in these two transmitter systems.

Animals↗

PubMed1989

Effects of parathyroid hormone on cyclic-AMP concentrations of in vitro Necturus maculosus gastric antrum.

The effects of bovine parathyroid hormone (bPTH1-84) on the stimulation of intracellular cyclic-AMP [cAMP] were investigated in an in vitro preparation of Necturus maculosus antral mucosa. When the antrum was exposed to 1, 5, 10, or 100 nM bPTH1-84, there was an approximately 2-fold nonlinear increase in tissue [cAMP] over basal values. The pretreatment of the antral mucosa with 1 mM isobutylmethylxanthine (IBMX, a phosphodiesterase inhibitor) increased with detectability of mucosal [cAMP]. The addition of 1, 5, 10, or 100 nM bPTH1-84 to tissues pretreated with IBMX resulted in an approximately 3.5-fold linear increase in mucosal [cAMP] over basal values. The time course of the generation of mucosal cAMP to 10 nM bPTH1-84 resulted in a small but significant transient increase at 2.5 min after the addition of bPTH1-84 but no change in the medium [cAMP]. In tissues pretreated with 1 mM IBMX the response to 10 nM bPTH1-84 was a large biphasic increase of [cAMP] at 2.5 min that progressively declined to near basal values by 15 min. There was also a significant sustained increase in the [cAMP] in the bathing medium at 2.5 min of tissues pretreated with IBMX followed by 10 nM bPTH1-84. These results suggest the presence of an adenylate cyclase that can be activated by a mammalian bPTH1-84 in elevating intracellular cAMP levels in the N. maculosus antral mucosa.

1-Methyl-3-isobutylxanthine↗

PubMed2003

Hepatic metallothioneins in two neotenic salamanders, Proteus anguinus and Necturus maculosus (Amphibia, Caudata).

The presence of metallothionein (MT) and the subcellular distribution of copper, zinc and cadmium were investigated in livers of two neotenic salamanders, Proteus anguinus and Necturus maculosus. In P. anguinus, caught in the wild, hepatic MTs were present as a single isoform of (Zn, Cu, Cd)-thioneins, whose molecular weight was estimated to be approximately 12000 by size exclusion chromatography. The percentage of zinc and cadmium was higher in the cytosol and of copper in the pellet. Cytosolic cadmium was almost exclusively associated with MTs (80%), while zinc and copper were also present in the regions of higher-molecular weight proteins. In laboratory bred N. maculosus, MTs were isolated from the liver cytosol and extract of the pellet as (Cu, Zn)- and (Zn, Cu)-thioneins, respectively. According to the low amount of copper extracting from liver pellets of N. maculosus, the presence of water insoluble aggregated forms of Cu-thioneins should be checked in further investigations.

Animals↗

PubMed1982

A micro-electrode study of oligopeptide absorption by the small intestinal epithelium of Necturus maculosus.

1. The effects of some amino acids and oligopeptides on the electrical properties of the brush-border membrane of the small intestine of the mudpuppy, Necturus maculosus were studied in vitro using micro-electrodes. 2. A number of amino acids (glycine, L-proline and L-leucine) and small peptides (carnosine, glycyl-L-proline, L-leucyl-L-leucine, glycylglycine and glycylglycylglycine) depolarized the brush-border membrane. This was associated with a reduction of input resistance. Tetraglycine did not appreciably reduce the membrane potential. 3. Evidence is presented that the electrical effects caused by application of the peptides are not solely due to the effects of the products of peptide hydrolysis. Furthermore there appears to be more than a single system available to the peptides. 4. Stereospecificity is found for both amino acids and peptides but appears to be more marked for the peptides. 5. Peptide-induced depolarizations are less markedly reduced in the absence of external Na+ than are the depolarizations caused by the amino acids. 6. These results are discussed with respect to the mechanism and significance of amino acid and peptide transport in the intestinal tract.

Amino Acids↗

PubMedDate not supplied

Pedomorphosis revisited: thyroid hormone receptors are functional in Necturus maculosus.

Heterochrony, a difference in developmental timing, is a central concept in modern evolutionary biology. An example is pedomorphosis, retention of juvenile characteristics in sexually mature adults, a phenomenon largely represented in salamanders. The mudpuppy (Necturus maculosus) is an obligate pedomorphic amphibian, never undergoing metamorphosis. Thyroid hormone induces tissue transformation in metamorphosing species and this action is mediated by nuclear thyroid hormone (TH) receptors (TRs). The absence of metamorphosis in Necturus has been attributed to a resistance to TH action as treatment with exogenous TH fails to induce transformation. The failure to metamorphose could be due to the lack of TR expression in target tissues, or to a loss of TR function. Toward understanding the molecular basis for the failure of Necturus tissues to respond to TH, and the ultimate cause for the expression of the obligate pedomorphic life history, we characterized the structure, function, and expression of TR genes in Necturus. Strikingly, we found that Necturus TRalpha and TRbeta genes encode fully functional TR proteins. These TRs bind both DNA and TH and can transactivate target genes in response to TH. Both TRalpha and TRbeta are expressed in various tissues. TH treatment in vivo induced expression in the gill of some but not all genes known to be activated by TH in anuran larvae, caused whole organism metabolic effects, but induced no external morphological changes in adults or larvae. Thus, Necturus possesses fully functional TRs and its tissues are not generally resistant to the actions of TH. Rather, the absence of metamorphosis may be due to the loss of TH-dependent control of key genes required for tissue transformation.

Animals↗

PubMed1991

Water permeability of ventricular cell membrane in choroid plexus epithelium from Necturus maculosus.

1. The osmotic water permeability Lp and the relations between the flows of H2O, K+ and Cl- were studied in the ventricular membrane of the epithelium from the choroid plexus of Necturus maculosus. 2. The flows were induced by abrupt changes in external osmolarity of the ventricular solution. Solution changes were convective and no effects of unstirred layers could be detected on measured parameters. 3. The initial rate of change in intracellular concentrations of K+ and Cl- was monitored by double-barrelled ion-selective microelectrodes. 4. The initial rate of flux of H2O could be monitored as the changes in the concentration of intracellular choline ions (Ch+i). When 0.5 mmol l-1 of choline chloride was added to the external solutions, Ch+i attained values of 1-5 mmol l-1. The dilution or concentration of Ch+i could be recorded by K+ electrodes since the sensitivity of these to Ch+ is more than 50 times greater than to K+. 5. The Lp of the ventricular membrane of the epithelium was 1.4-2.1 x 10(-4) cm s-1 (osmol l-1)-1 and independent of the direction of the induced water flux. Lp was unchanged in tissues adapted to osmolarities of half the physiological value. 6. The efflux of H2O induced by mannitol was associated with an instantaneous efflux of K+ which was inhibited by furosemide. The fluxes had a ratio of 40 mmol l-1. The influx of H2O induced by the removal of NaCl from the ventricular solution was associated with an instantaneous influx of K+. The H2O influx had a ratio to the flux of K+ of 70 mmol l-1. 7. The efflux of H2O induced by mannitol was associated with an efflux of Cl- which was inhibited by furosemide. The ratio of the two fluxes was in the range 15-44 mmol l-1. 8. The conclusion is that the Ch+ method gives a reliable measure of the movement of H2O across the ventricular membrane. The magnitude of the Lp and its relevance to transepithelial transport are discussed. The osmotically induced H2O movement is accompanied by furosemide-sensitive fluxes of K+ and Cl- of the same magnitude. This suggests that co-transport between H2O and KCl can take place in the membrane.

Animals↗

PubMed1977

The permeability of the skin of a neotenous urodele amphibian, the mudpuppy Necturus maculosus.

1. The permeability of the isolated skin of a neotenous urodele amphibian, the mudpuppy Necturus maculosus, to Na, Cl, urea and water was measured. 2. Unidirectional transcutaneous flux measurements and the action of ouabain and amiloride, showed that there was normally no active Cl or Na transport, nor a Cl/Cl exchange diffusion process. 3. Amphotericin B initiated a transcutaneous potential difference and short-circuit current, which could be inhibited by ouabain. 4. The short-circuit current was nearly equivalent to the net Na Transport and this was also inhibited by ouabain. 5. A transcutaneous active Na transport mechanism thus appears to be incipient in the mudpuppy but is limited by a low permeability of the outer barrier of the cells. 6. Vasotocin increased the skin's diffusion permeability for water but had no effect on the influx of Na or urea. 7. The function of Necturus skin is in several respects unique compared to that of other amphibians.

Amiloride↗

PubMed2002

Two second messengers mediate amino acid responses in olfactory sensory neurons of the salamander, Necturus maculosus.

Odor transduction mediated by the adenylyl cyclase/cAMP pathway has been well studied, but it is still uncertain whether this pathway mediates the transduction of all odors in vertebrates. We isolated olfactory sensory neurons from the salamander Necturus maculosus and used calcium imaging with the indicator dye fura-2 to examine olfactory responses elicited by amino acids. The properties of approximately two-thirds of the odor responses suggested they were mediated by the adenylyl cyclase/cAMP pathway, but one-third of the responses were not mimicked by cAMP analogs nor blocked by inhibition of adenylyl cyclase, suggesting that these odor responses were mediated differently. Responses that were unaffected by inhibition of adenylyl cyclase were blocked by neomycin, an inhibitor of phospholipase C, implying that they were transduced by activation of phospholipase C. Some cells which responded to more than one amino acid appeared to employ both pathways, but each was used to transduce different odors. In addition, many responses that were mediated by the adenylyl cyclase/cAMP pathway were enhanced following inhibition of phospholipase C, suggesting that the phospholipase C pathway has a role not only in odor transduction, but also in the modulation of olfactory responses.

Action Potentials↗

PubMed1987

Ultrastructure of apical specializations of taste cells in the mudpuppy, Necturus maculosus.

The first interaction of taste stimuli with lingual chemoreceptors occurs on the apical membrane of taste cells, since only that portion is exposed to the oral cavity. To gain better insight into this interaction, we examined the pore region of taste buds in Necturus maculosus with scanning electron microscopy (SEM), transmission electron microscopy, and high-voltage electron microscopy. SEM of the pore reveals a patchwork distribution of three morphologically distinct types of apical specializations: long and branched (LB) microvilli, short and unbranched (SU) microvilli, and bundles of stereocilia. As demonstrated in thin and thick sections, LB microvilli are specializations of dark cells, SU microvilli are the apical specializations of light cells, and stereocilia arise from a cell that has the cytoplasmic markers characteristic of light cells. When left in place, the pore mucus completely covers the SU microvilli and partially covers the LB microvilli. However, stereocilia project above the surface and thus are highly exposed to taste stimuli in the oral cavity. These three morphologically distinct types of apical specializations may reveal functional differences among taste cells. The initial interaction between chemical stimulus and taste cell, and possibly chemoreceptor specificity itself, may be influenced by the morphology of the apical ending.

Animals↗

PubMed1992

Influence of temperature and photoperiod on plasma melatonin in the mudpuppy, Necturus maculosus.

A melatonin (MEL) radioimmunoassay employed previously only in mammals was used to estimate plasma MEL in a salamander, Necturus maculosus. Validation procedures included thin-layer chromatography of plasma extracts, parallel inhibition curves of authentic MEL and serially diluted plasma and plasma extracts and quantitative recovery of authentic MEL added to pooled Necturus plasma. A diel cycle of plasma MEL was demonstrated in mudpuppies acclimatized for a minimum of 3 weeks under a 12L:12D photoperiod and 15 +/- 1 degrees and sampled in late March. The MEL cycle persisted under a reversed photoperiod (lights on, 1800 hr), but the amplitude of the MEL peak was diminished, and the peak was more than 180 degrees out of phase with the corresponding peak under a normal photoperiod. In animals acclimated to 5 degrees in mid-June under a 12L:12D photoperiod, the diel cycle of plasma MEL continued, but both midphotophase and midscotophase concentrations were reduced compared with 15 degrees controls. The diel cycle was also present in animals acclimated to 25 degrees, but the decrease in MEL concentrations was less marked than that in 5 degrees animals. Photoperiod is apparently the primary cue for cycles in plasma MEL in Necturus, but the cycle can be influenced by temperature.

Animals↗