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PubMed1982

Roles of the skin and gills in sodium and water exchanges in neotenic urodele amphibians.

Neotenic urodele amphibians that possess prominent external gills absorb substantial amounts of water and sodium across their integument. Ligation of the gills did not change the uptake of either water or sodium in Necturus maculosus or Ambystoma tigrinum. In anuran tadpoles and fish the gills are known to be a major site of such exchanges. The possible physiological significance of such a restricted branchial permeability in aquatic urodeles is discussed as well as the bases for these special properties.

Ambystoma↗
PubMed2006

Form and function of ON-OFF amacrine cells in the amphibian retina.

ON-OFF amacrine cells were studied with whole cell recording techniques and intracellular staining methods using intact retina-eyecup preparations of the tiger salamander (Ambystoma tigrinum) and the mudpuppy (Necturus maculosus). Morphological characterization of these cells included three-dimensional reconstruction methods based on serial optical sections obtained with a confocal microscope. Some cells had their detailed morphology digitized with a computer-assisted tracing system and converted to compartmental models for computer simulations. The dendrites of ON-OFF amacrine cells have spines and numerous varicosities. Physiological recordings confirmed that ON-OFF amacrine cells generate both large- and small-amplitude impulses attributed, respectively, to somatic and dendritic generation sites. Using a multichannel model for impulse generation, computer simulations were carried out to evaluate how impulses are likely to propagate throughout these structures. We conclude that the ON-OFF amacrine cell is organized with multifocal dendritic impulse generating sites and that both dendritic and somatic impulse activity contribute to the functional repertoire of these interneurons: locally generated dendritic impulses can provide regional activation, while somatic impulse activity results in rapid activation of the entire dendritic tree.

Amacrine Cells↗
PubMed1998

Mechanics of lung ventilation in a large aquatic salamander, siren lacertina

Lung ventilation in Siren lacertina was studied using X-ray video, measurements of body cavity pressure and electromyography of hypaxial muscles. S. lacertina utilizes a two-stroke buccal pump in which mixing of expired and inspired gas is minimized by partial expansion of the buccal cavity during exhalation and then full expansion after exhalation is complete. Mixing is further reduced by the use of one or two accessory inspirations after the first, mixed-gas cycle. Exhalation occurs in two phases: a passive phase in which hydrostatic pressure and possibly lung elasticity force air out of the lungs, and an active phase in which contraction of the transverse abdominis (TA) muscle increases body cavity pressure and forces most of the remaining air out. In electromyograms of the lateral hypaxial musculature, the TA became active 200-400 ms before the rise in body cavity pressure, and activity ceased at peak pressure. The TA was not active during inspiration, and no consistent activity during breathing was noted in the external oblique, internal oblique and rectus abdominis muscles. The finding that the TA is the primary expiratory muscle in S. lacertina agrees with findings in a previous study of another salamander, Necturus maculosus. Together, these results indicate that the use of the TA for exhalation is a primitive character for salamanders and support the hypothesis that the breathing mechanism of salamanders represents an intermediate step in evolution between a buccal pump, in which only head muscles are used for ventilation, and an aspiration pump, in which axial muscles are used for both exhalation and inhalation.

Journal Article↗
PubMed1977

Retina viability in the Necturus.

Electroretinograms (ERG's) were recorded from the eyes of paralyzed normal and decapitated mudpuppies (Necturus maculosus). Fully dark-adapted responses to a range of stimulus intensities were compiled hourly under both conditions. Statistical analyses indicate no significant change in responsiveness in paralyzed normal animals during periods up to 6 hours. After decapitation, there was a significant decline of sensitivity to light during the first 3 hours. An oxygen-sensitive component similar to the c-wave was observed with DC recording but only in normal eyes. Anoxic mudpuppy eyes show an ERG decline with an average time constant of 1.5 hours.

Animals↗
PubMed1997

Thyroid hormone receptors in perennibranchiate amphibians.

Thyroid hormone has long been known to induce metamorphosis in amphibians. The understanding of the molecular steps controlling the completion of metamorphosis has nevertheless been hampered by the complexity of this event. The comparison of organisms in which metamorphosis does or does not occur, may provide clues into the molecular cascade that control it. Up to now the available data suggest that perennibranchiate amphibians retain their larval characters mainly because their tissues do not respond to thyroid hormones. In such a context the recent identification of a thyroid hormone receptor alpha in the perennibranchiate Proteus anguinus is provocative (Ho Huynh et al., Int. J. Dev. Biol. 40:537-543, 1996). In the present paper, we provide evidences that this recently described sequence is in fact a sequence from Xenopus laevis. Indeed, we identified the authentic thyroid hormone receptors of both alpha and beta types in two perennibranchiate species Necturus maculosus and Proteus anguinus. The various controls required to ascertain the authenticity of a developmental gene cloned by PCR or RT-PCR analysis are presented. The results reported in the present paper are relevant with phylogenetical analysis. This induces our team to conclude that the Proteus TR alpha sequence reported by Ho Huynh et al. (1996) reflects a contamination of the RT-PCR by Xenopus laevis material.

Amphibians↗
PubMed1991

Further studies of electrogenic Na+/HCO3- cotransport in glial cells of Necturus optic nerve: regulation of pHi.

In the presence of Ba++, an increase in the bath HCO3- at constant CO2 (i.e., variable bath pH) produced a hyperpolarization. The hyperpolarizing effect of adding HCO3-/CO2 at constant bath pH was not significantly affected by the presence of 50 mumol/l strophanthidin. In the absence of Ba++, addition of HCO3-/CO2 at constant bath pH produced a Na(+)-dependent hyperpolarization. Therefore, CO2 movements, electrogenic Na+/K+ pump activity and changes in Ba++ binding do not contribute significantly to the hyperpolarization induced by HCO3-. These results along with the results of previous studies (Astion et al: J Gen Physiol 93:731, 1989) strongly suggest that the hyperpolarization induced by the addition of HCO3- is due to an electrogenic Na+/HCO3- cotransporter, which transports Na+, HCO3- (or its equivalent), and net negative charge across the glial membrane. To study the role of electrogenic Na+/HCO3- cotransport in the regulation of pHi in glial cells, we used intracellular double-barreled, pH-sensitive microelectrodes. At a bath pH of 7.5, the mean initial intracellular pH (pHi) was 7.32 (SD 0.03, n = 6) in HEPES-buffered Ringer's solution and 7.39 (SD 0.1, n = 6) in HCO3-/CO2 buffered solution. These values for pHi are more than 1.2 pH units alkaline to the pHi predicted from a passive distribution of protons; thus, these cells actively regulate pHi. Superfusion and withdrawal of 15 mmol/l NH4+ induced an acidification of 0.2 to 0.3 pH units, which recovered toward the original steady-state pHi.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Necturus gallbladder epithelial cell volume regulation and inhibitors of arachidonic acid metabolism.

Inhibition of the metabolism of arachidonic acid by the epoxygenase (cytochrome P-450) pathway with the inhibitor ketoconazole results in excessive cell swelling upon exposure to hyposmolality instead of the rapid and complete regulatory volume decrease (RVD) normally observed. NaCl entry from bathing solutions to cell interior was shown to cause this swelling, with Na influx occurring across the basolateral membrane and electrically silent Cl influx across the apical membrane. Ion substitution experiments show that the KCl efflux mediating RVD was unimpaired by ketoconazole, but was overwhelmed by the NaCl influx. Measurements of transepithelial fluid flux, Cl concentration, osmolality and pH showed that gallbladders treated with ketoconazole transiently secreted fluid rather than the normal absorption. We conclude that inhibition of arachidonic acid metabolism does not directly affect RVD by Necturus gallbladder, but that blockade of the epoxygenase pathway can have a profound influence on NaCl entry into gallbladder epithelial cells.

Animals↗
PubMed1989

Anion channels in a leaky epithelium. A patch-clamp study of choroid plexus.

We have used the patch-clamp technique to characterize three anion channels in the ventricular membrane of the choroid plexus epithelium from Necturus. The most frequently occurring channel had a nonlinear IV-curve. The conductance in excised patches with 112 mM chloride at both sides was 28 pS at 0 mV, increasing towards positive membrane potentials. The selectivity ratios were PNa:PCl less than or equal to 0.1 and PNO3:PCl:PHCO3 = 1.6:1:0.43. SITS and furosemide (1 mM) on the inside reduces chloride flux to 0.15 and 0.37 times the control value. In attached patches, the most commonly observed channel had a conductance of 7.5 pS. The single-channel current for this channel reversed direction at 15 mV hyperpolarization, indicating accumulation of chloride to a factor of 1.8 above equilibrium. External stimulation of the tissue by theophylline, IBMX and dbcAMP, or by hypotonic shock did not increase the activity of this channel. In very few excised patches, we have observed a chloride channel with a conductance of 7 pS with 112 mM chloride at both sides. The 7 pS channel appears to be identical to a 2 pS channel found in attached patches. The 2 pS channel was not normally active in attached patches but was activated in 28% of the patches by external stimulation. Finally, in few excised patches we have found a 375 pS channel which inactivates within seconds when membrane potential is stepped from 0 mV to a value that differs more than 10-20 mV from zero. The channel did not conduct gluconate but PNO3:PCl = 1.08 and PNa:PCl less than or equal to 0.1. Internal SITS and furosemide (1 mM) reduced chloride flux to 0.3 and 0.5 times the control value. The channel was never seen in attached patches. The current carried through these channels can not account for the transepithelial steady state Cl- -flux measured by microelectrodes. KCl exit from the cell is suggested to be carried by KCl-cotransport or by channels that are too small to be seen in patch-clamp experiments.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Calcium is not involved in the cAMP-mediated stimulation of Cl- conductance in the apical membrane of Necturus gallbladder epithelium.

The permeability properties of the forskolin-stimulated Cl- conductance in the apical membrane of Necturus gallbladder epithelium and the possible participation of intracellular Ca2+ in its stimulation have been investigated. The anion selectivity sequence as derived from biionic potential measurements (SCN- > I- approximately NO3- > Br- > Cl- >> ISE-) differed from the sequence derived from measurements of apical membrane resistance (NO3- approximately Br- approximately Cl- > SCN- > I- approximately ISE-). Accordingly, the conductance was inhibited by SCN- and I- which, from the potential measurements, appeared to be more permeable than Cl-. This finding agrees with observations of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel reported recently. However, none of the commonly used Cl- channel blockers, such as 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), anthracene-9-carboxylic acid (9-AC) and glibenclamide reduced this conductance in Necturus gallbladder. In contrast to the situation in most other epithelia, elevation of intracellular Ca2+ concentration ([Ca2+]i) by ionomycin stimulated only K+ conductance and not that of Cl- in the apical cell membrane. Chelation of intracellular Ca2+ did not prevent the stimulation of Cl- conductance by forskolin. This indicates that [Ca2+]i does not have even a permissive role in the cyclic adenosine monophosphate-(cAMP)-mediated stimulation process, as would have been expected if exocytosis was involved. Further evidence against the involvement of exocytosis in the stimulation process came from the observation that the stimulation was not associated with an increase in apical membrane capacitance and was not suppressed by disruption of the cytoskeleton by preincubation of the tissue with cytochalasin D. The data indicate that Necturus gallbladder epithelium contains homologues of the CFTR Cl- channel which reside permanently in the apical cell membrane and which can be stimulated by a cAMP-dependent phosphorylation process without involvement of cell Ca2+ or exocytosis.

Animals↗
PubMed1993

Tight-junction tightness of Necturus gall bladder epithelium is not regulated by cAMP or intracellular Ca2+. I. Microscopic and general electrophysiological observations.

Following the publications by Duffey et al. [Nature 294:451 (1981)] and Palant et al. [Am J Physiol 245: C203 (1983)] it is generally accepted that tight-junction tightness of Necturus gall bladder epithelium is up-regulated by cAMP-mediated and Ca(2+)-mediated stimulation. This conclusion was mainly based on observed increases in transepithelial resistance (Rt). However, since in leaky epithelia Rt cannot be simply equated with the tight junction resistance (Rj), but may include large contributions from the lateral space resistance (Rlis), we asked whether the observed increases in Rt resulted indeed from Rj or whether Rlis also increased. The experiments were performed on Necturus gall bladders using forskolin or the Ca2+ ionophore A23187 as stimulants. Forskolin (2 mumol/l) had a biphasic effect. In the first 5 min Rt decreased from 128 +/- 13 to 119 +/- 14 omega cm2 (P < 0.05, n = 10) which probably reflects stimulation of an apical cell membrane Cl- conductance (see accompanying paper). Subsequently Rt increased in approximately 30 min to 184 +/- 20 omega cm2 and then remained fairly constant. Simultaneously the lateral spaces collapsed. If the spaces were now transiently opened by passing mucosa-positive direct current across the epithelium, Rt fell transiently to 111 +/- 7 omega cm2, but returned gradually to its elevated level when the spaces collapsed again. When the spaces were constantly dilated by a serosa-positive hydrostatic pressure of 1 cm H2O, forskolin neither affected the space width nor increased Rt, and current passage was virtually ineffective, although the cells depolarized in response to forskolin as usual.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Tight-junction tightness of Necturus gall bladder epithelium is not regulated by cAMP or intracellular Ca2+. II. Impedance measurements.

In the preceding publication we have reported that, contrary to the prevailing opinion in the literature, the tight-junction tightness of Necturus gall bladder epithelium is not up-regulated by cAMP-mediated or by Ca(2+)-mediated stimulation. This conclusion was based on our observation that the stimulant-induced increase in transepithelial resistance (Rt) occurred only when the lateral intercellular spaces were allowed to collapse, which suggested that the increase reflected primarily or exclusively the increasing resistance of the lateral spaces (Rlis) rather than the postulated increase in tight-junction resistance (Rj). An alternative explanation could have been that the constancy of Rt after space dilatation reflected an increase Rj that was masked by a concomitant fall in apical and basolateral cell membrane resistances Ra and Rbl. To decide between those possibilities we have performed impedance measurements with transepithelial and intracellular microelectrodes on Necturus gall bladder epithelium. Applying previously developed analysis procedures, the measurements readily showed that elevation of intracellular Ca2+ concentration increased Rlis, but left Rj as well as Ra and Rbl quasi constant. Experiments with forskolin, theophylline or isobutylxanthine, on the other hand, were less clear. These stimulants activated an apical Cl- conductance, which drastically reduced Ra and apparently caused low-frequency polarization effects that could not be accounted for by the classical epithelial equivalent circuit. After elimination of the polarization phenomena by uni- or bilateral substitution of Cl- by isethionate or sulphate, however, we were able to demonstrate that Rj remains constant under cAMP-mediated stimulation irrespective of whether the lateral spaces are kept open or are allowed to collapse. We conclude that the tight-junction resistance of Necturus gall bladder epithelium is not controlled by intracellular Ca2+ or by cAMP-mediated stimulation.

Animals↗
PubMed1985

Electrophysiological studies on lateral intercellular spaces of Necturus gallbladder epithelium.

The lateral intercellular spaces of Necturus gallbladder epithelium were punctured with double-barrelled ion selective microelectrodes in order to determine the ion concentrations of lateral space fluid and the contribution of the lateral spaces to transepithelial resistance. Neither under control conditions, nor after diluting the bathing fluids to increase the rate of volume absorption, nor during passage of direct current of 200 microA/cm2, were any reliable concentration differences observed between lateral space fluid and external bathing fluids. These observations suggest that water can follow salt transport without requiring osmotic concentration gradients of greater than 1 or 2 mosmol/l and indicate that recently observed high values of water permeability must still be considered as underestimates. After developing a test to recognize and exclude leaky punctures, the contribution of the lateral spaces to transepithelial resistance could be determined. It amounted to around 29%. This value agrees well with results from recent impedance measurements which were performed under control conditions in the same preparation.

Animals↗
PubMed1985

On the luminal membrane permeability to Cl- of necturus gastric surface cells.

The surface cells (SEC) luminal membrane conductance to Cl- was tested on the resting Necturus gastric mucosa by applying conventional and Cl--sensitive microelectrodes under luminal low-Cl- conditions. Reduction of chloride (2 mmol/1) in the luminal bath did not result in any detectable reduction of intracellular Cl-. Therefore, in analogy to previous findings on the frog SEC, also the luminal membrane of the Necturus surface cells appears to have a negligible conductance to Cl-. The data speak against a relevant role of the amphibian SEC in the non-acidic Cl- secretion.

Animals↗
PubMed1984

Rapid determination of intraepithelial resistance barriers by alternating current spectroscopy. I. Experimental procedures.

A method has been developed to determine rapidly and simultaneously the resistance of the tight junctions, the resistance of the lateral intercellular space, and the resistances and capacitances of the apical and basolateral cell membrane in leaky epithelia by alternating current spectroscopy. The present paper describes the experimental procedures. Multi-frequency sine-wave currents are simultaneously applied across the tissue and the transepithelial and intracellular voltage responses are recorded with shielded microelectrodes, digitized, and stored in a computer. Using Fast Fourier Transform techniques the frequency-dependent transepithelial impedance and an apparent basal cell membrane impedance are then calculated from the voltage and current waveform. By fitting appropriate model circuits to the data the above listed individual resistances can be deduced. The model calculations and the fit results are described in the subsequent paper [14]. In the present design the analysis covers the frequency range between 2.5 Hz and 12.5 kHz. The minimal measuring time is in the order of 1-2s.

Animals↗
PubMed1984

Rapid determination of intraepithelial resistance barriers by alternating current spectroscopy. II. Test of model circuits and quantification of results.

The impedance of Necturus gallbladder epithelium was measured with transepithelial and intracellular microelectrodes in different transport states. The data are analysed with five electrical equivalent circuits, which differ with respect to the configuration of the paracellular shunt path (lumped vs distributed model of the lateral space), and of the apical cell membrane (non-ideal capacitance or surface amplification by micro-tubular infoldings). Least square fits indicate: 1. that the lumped model cannot represent the epithelium properly, even under control conditions; 2. that the distributed model, which considers the lateral intracellular space separately as a cable-like structure, describes the data well, both under control conditions and during collapse of the lateral spaces; and 3. that the above indicated variations of the apical membrane configuration improve the fits, but have little effect on the magnitude of the calculated circuit parameters. Quantitatively the analysis of 214 measurements on 25 gallbladders under control conditions yields the following results: The resistances of the tight junctions, of the lateral intercellular space, and of the apical and basal cell membrane are Rj = 123, Rlis = 35.5, Ra approximately 3,500, and Rb = 225 (all in omega cm2), and the capacitances of the cell membranes are Ca = 4.95 and Cbl = 26.5 (mu F/cm2). In oxygen deficiency and after cessation of chamber perfusion transport decreased, the spaces collapsed, and Rlis increased to approximately 130 omega cm2. Although the accuracy of the estimated Ra values is still limited, the analysis shows that it is possible to determine Rj and Rlis separately and to follow their changes in response to experimental maneuvers.

Animals↗
PubMed1986

Potassium induced changes in cell volume of gallbladder epithelium.

The mechanisms of transmembrane K and anion movements were investigated by measurement of the changes in cell volume, apical membrane potential difference, and intracellular K activity resulting from exposure of Necturus gallbladder to solutions with increased K concentration. Cell swelling occurred when the tissue was exposed bilaterally to 25 mmol/l K. This swelling was both Cl and HCO3 dependent, but was not blocked by DIDS or bumetanide. Unilateral tenfold increases in extracellular K concentration did not cause cell swelling; addition of 5 mmol/l Ba to the contralateral cell surface resulted in cell volume increases comparable to those seen with bilateral K increase. Complete blockage of K channels by Ba could be demonstrated electrophysiologically at normal extracellular K concentrations but not in the presence of increased K. Our results were consistent with the passive movement of K through Ba-sensitive channels in both cell membranes. We were unable to detect other mechanisms for transmembrane K movement. The cell swelling caused by exposure to 25 mmol/l K was not due to intracellular K accumulation and may be related to the effects of membrane depolarization on voltage sensitive anion transport processes.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1982

Cellular and paracellular resistances of the Necturus proximal tubule.

Individual resistances of the apical cell membrane Ra, the basolateral cell membrane, Rbl, and the paracellular shunt, Rs, were determined in the Necturus proximal tubule using a set of three electrical parameters. Four electrical parameters were measured: the transepithelial resistance, (Rte), the apical and basolateral cell membrane resistance in parallel, (Rz free-flow tubules), the basolateral cell membrane resistance in oil-filled tubules, (Rz oil-filled), and the ratio of apical and basolateral cell membrane resistance (Ra/Rbl). Rte was determined from an analysis of the spatial decay of luminal voltage following luminal current injection. Rz free-flow and Rz oil-filled were measured by the analysis of the spatial decay of intracellular voltage deflections following cellular current injection in free flow and oil-filled tubules, respectively. Ra/Rbl was estimated from the ratio of voltage deflections across the apical and basolateral cell membranes following transepithelial current injection. In addition, the magnitude of cellular and luminal cable interactions was evaluated, by comparing the spatial decay of voltage deflections in the cell and in the lumen following intracellular current injection. The combined cell membrane resistance (Ra + Rbl) is between one to two orders of magnitude greater than the paracellular resistance. This result supports the view that the Necturus proximal tubule is a leaky epithelium.

Animals↗