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PubMed1990

Chemotransduction in Necturus taste buds, a model for taste processing.

The taste bud in Necturus serves as a good model for taste mechanisms in vertebrates. The large size of taste cells and relative accessibility of the tissue for detailed electrophysiological and ultrastructural studies makes this species well-suited for studying taste transduction. Important features of taste transduction that have been learned from investigations in Necturus are that voltage-gated potassium channels are preferentially distributed on the apical membrane of taste cells; voltage-gated potassium channels allow K ions to enter the cell when taste buds are stimulated with K salts; some chemical stimuli act by closing K channels, thereby eliciting depolarizing receptor potentials in taste cells. Many of these findings have been confirmed and extended in other animals, including mammals. Furthermore, recent evidence from experiments in Necturus suggests that there is a considerable degree of synaptic coupling among taste cells. This synaptic coupling could form the basis for signal processing and integration in the peripheral sensory organs of taste, the taste buds.

Animals↗
PubMed1989

Monoclonal antibodies to the apical chloride channel in Necturus gallbladder inhibit the chloride conductance.

Monoclonal antibodies raised by injecting Necturus gallbladder cells into mice were tested for their ability to inhibit the apical chloride conductance induced by elevation of cellular cAMP. Five of these monoclonal antibodies bound to the apical cells, as shown by indirect immunofluorescence microscopy, and inhibited the chloride conductance; one antibody that bound only to subepithelial smooth muscle, by indirect immunofluorescence microscopy, showed no inhibition of chloride transport. The channel or a closely related molecule is present in the membrane whether or not the pathway is open, since, in addition to inhibiting the conductance of the open channel, the antibody also bound to the membrane in the resting state and prevented subsequent opening of the channel. The antibody was shown to recognize, by ELISA, epitopes from the Necturus gallbladder and small intestine. Finally, by Western blot analysis of Necturus gallbladder homogenates, the antibody was shown to recognize two protein bands of Mr 219,000 and Mr 69,000. This antibody should permit isolation and characterization of this important ion channel.

Animals↗
PubMed1988

A chloride conductance activated by adenosine 3',5'-cyclic monophosphate in the apical membrane of Necturus enterocytes.

1. Intracellular potentials, Cl- activity and membrane resistances were measured in Necturus small intestinal epithelium during Cl- replacement experiments using conventional or Cl- -selective double-barrelled microelectrodes. A Cl- conductance, located in the apical membrane and activated by cyclic nucleotides is demonstrated by ion-substitution experiments. 2. The mean mucosal membrane potential (Em) was -35.5 mV. Removal of Cl- from the mucosal medium by replacement with gluconate, evoked a sudden depolarization of Em and an immediate increase in the fractional resistance of the mucosal membrane (f(Rm)). The size of the change in Em varied between 3 and 65 mV, corresponding to Cl- to K+ permeability ratios between 0.2 and 20. It was inversely related to the initial f(Rm), which ranged from 0.04 to 0.50. 3. Prolonged incubation in low-Cl- solutions led to a reversal of the initial depolarization and to a sustained hyperpolarization accompanied by a marked increase in f(Rm). The new value of Em was close to the K+ equilibrium potential, consistent with a depletion of cellular Cl- and the preponderance of a K+ membrane permeability in the absence of Cl-. This emphasizes the role of Cl- in establishing Em. 4. Removal of mucosal Cl- produced a fast decrease in intracellular Cl-, as measured with Cl- -selective microelectrodes. The efflux was consistent with electrodiffusion across the mucosal membrane. Changes in Em paralleled changes in intracellular Cl- activity, indicating the presence of a large Cl- conductance. 5. Dibutyryl cyclic AMP or forskolin produced a slow depolarization, a decrease in f(Rm) and an increased change in intracellular potential in low mucosal Cl- which on average corresponds to an approximately 15-fold increase in the relative Cl- permeability. These results are consistent with an activation of apical Cl- conductance. 6. The selectivity of Cl- channels of Necturus enterocytes to different anions was obtained from potential measurements. The sequence of permeabilities was SCN- greater than I- greater than or equal to Br- greater than NO3- greater than Cl- much greater than HCO3- greater than gluconate. This is consistent with a model involving a weak interaction of the anions with the selectivity filter. 7. The selectivity of the anion conductance was maintained after activation with cyclic nucleotides, suggesting a single channel for the permeation of the different anions tested, rather than parallel channels. 8. Derivatives of 9-anthracene which are potent inhibitors of Cl- channels in other systems failed to block the apical Cl- conductance of Necturus enterocytes. Chloride conductance was also insensitive to furosemide.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Regulation of apical membrane ion transport in Necturus gallbladder.

Na and Cl movement through the apical membrane of Necturus gallbladder epithelium was investigated using electrophysiological and light microscopic measurements. Changes in membrane potential difference, fractional resistance of the apical membrane, and transepithelial resistance caused by changes in apical bath Cl concentration revealed the presence of a Cl conductance in the apical membrane of control tissues that was apparently not present in the preparations studied by other investigators. This Cl conductance was blocked by bumetanide (10(-5) M) or by the inhibitor of adenosine 3',5'-cyclic monophosphate (cAMP) action, the Rp isomer of adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS; 0.5 mM). Treatment of the tissues with Rp-cAMPS also eliminated bumetanide-sensitive cell swelling in the presence of ouabain and activated an amiloride-sensitive swelling, changes consistent with inhibition of NaCl cotransport and the activation of Na-H and Cl-HCO3 exchange. We conclude that the mode of NaCl entry into Necturus gallbladder epithelial cells is determined by the level of cAMP. When cAMP levels are high, entry occurs by NaCl cotransport; when cAMP levels are low, parallel exchange of Na-H and Cl-HCO3 predominates. These observations explain the previous disagreements about the mode of NaCl entry into Necturus gallbladder epithelial cells.

Animals↗
PubMed1993

Immunoisolation of a K+ channel from basolateral membranes of Necturus enterocytes.

We have reported that a peptide composed of the NH2-terminal 22 amino acids of the Drosophila Shaker B K+ channel protein, which is responsible for the inactivation of this A-type channel, blocks the inner, open mouth of a voltage-gated K+ channel present in the basolateral membrane of Necturus maculosa small intestinal enterocytes. We now demonstrate that antibodies to this "inactivating" peptide interact with proteins in solubilized and intact basolateral membranes from Necturus enterocytes. Asolectin vesicles reconstituted with the full complement of solubilized basolateral membrane proteins display 86Rb+ uptake that is inhibited by tetraethylammonium ion and abolished by immunoprecipitation with these antibodies. Furthermore, asolectin vesicles containing protein eluted from an antibody-affinity column display 86Rb+ uptake that is abolished by boiling. Finally, reconstitution of the immunoisolated protein into planar phospholipid bilayers disclosed a K+ channel whose single-channel properties are identical to those of the voltage-gated channel in the native basolateral membranes. Our data are consistent with the notion that a 150-kDa protein present in basolateral membranes of Necturus enterocytes possesses inwardly rectifying K+ channel activity and that this protein is antigenically similar to the type A K+ channel present in the flight muscles of Drosophila melanogaster and encoded by the Shaker B locus.

Animals↗
PubMed1997

Identification and functional assay of an extracellular calcium-sensing receptor in Necturus gastric mucosa.

In mammals and amphibians, increases in extracellular Ca2+ can activate bicarbonate secretion and other protective functions of gastric mucosa. We hypothesized that the recently cloned extracellular Ca(2+)-sensing receptor (CaR) is functioning in the gastric mucosa. In Necturus maculosus gastric mucosa, reverse transcription-polymerase chain reaction using primers based on previously cloned CaR sequences amplified a 326-bp DNA fragment that had 84% nucleotide sequence identity with the rat kidney CaR. Immunohistochemical localization of the CaR using specific anti-CaR antiserum revealed its presence on the basal aspect of gastric epithelial cells. In microelectrode studies of Necturus antral mucosa, exposure to elevated Ca2+ (4.8 mM) and the CaR agonists NPS-467 and neomycin sulfate resulted in significant hyperpolarizations of basal membrane electrical potentials and increases in apical-to-basal membrane resistance ratios. Circuit analysis revealed that these changes reflected specific decreases in basolateral membrane resistance. Inhibition of prostaglandin synthesis using indomethacin significantly attenuated these effects. We conclude that the CaR is present and functioning in Necturus gastric antrum.

Amiloride↗
PubMed1988

Ca2+-activated K+ channels in the apical membrane of Necturus choroid plexus.

The properties of Ca2+-activated K+ channels in the apical membrane of the Necturus choroid plexus were studied using single-channel recording techniques in the cell-attached and excised-patch configurations. Channels with large unitary conductances clustered around 150 and 220 pS were most commonly observed. These channels exhibited a high selectivity for K+ over Na+ and K+ over Cs+. They were blocked by high cytoplasmic Na+ concentrations (110 mM). Channel activity increased with depolarizing membrane potentials, and with increasing cytoplasmic Ca2+ concentrations. Increasing Ca2+ from 5 to 500 nM, increased open probability by an order of magnitude, without changing single-channel conductance. Open probability increased up to 10-fold with a 20-mV depolarization when Ca2+ was 500 nM. Lowering intracellular pH one unit, decreased open probability by more than two orders of magnitude, but pH did not affect single-channel conductance. Cytoplasmic Ba2+ reduced both channel-open probability and conductance. The sites for the action of Ba2+ are located at a distance more than halfway through the applied electric field from the inside of the membrane. Values of 0.013 and 117 mM were calculated as the apparent Ba2+ dissociation constants (KD(0 mV] for the effects on probability and conductance, respectively. TEA+ (tetraethylammonium) reduced single-channel current. Applied to the cytoplasmic side, it acted on a site 20% of the distance through the membrane, with a KD(0 mV) = 5.6 mM. A second site, with a higher affinity, KD(0 mV) = 0.23 mM, may account for the near total block of channel conductance by 2 mM TEA+ applied to the outside of the membrane. It is concluded that the channels in Necturus choroid plexus exhibit many of the properties of "maxi" Ca2+-activated K+ channels found in other tissues.

Animals↗
PubMed1984

Regulation of the basolateral potassium conductance of the Necturus proximal tubule.

Two methods, the measurement of the response of the basolateral membrane potential (Vbl) of proximal tubule cells of Necturus to step changes in basolateral K+ concentration, and cellular cable analysis, were used to assess the changes in basolateral potassium conductance (GK) caused by a variety of maneuvers. The effects of some of these maneuvers on intracellular K+ activity (aiK) were also evaluated using double-barreled ion-selective electrodes. Perfusion with 0 mM K+ basolateral solution for 15 min followed by 45 min of 1 mM K+ solution resulted in a fall in basolateral potassium (apparent) transference number (tK), Vbl and aiK. Results of cable analysis showed that total basolateral resistance, Rb, rose. The electrophysiological effects of additional manipulations, known to inhibit net sodium reabsorption across the proximal tubular epithelium of Necturus, were also investigated. Ouabain caused a fall in tK accompanied by large decreases in aiK and Vbl. Lowering luminal sodium caused a fall in tK and a small reduction in Vbl. Selective reduction of peritubular sodium, a maneuver that has been shown to block sodium transport from lumen to peritubular fluid, also resulted in a significant decrease in tK. These results suggest that GK varies directly with rate of transport of the sodium pump, irrespective of the mechanism of change in pump turnover.

Animals↗
PubMed1989

Measurement of the effective thickness of the mucosal unstirred layer in Necturus gallbladder epithelium.

The effective thickness of the unstirred fluid layer (USL) adjacent to an epithelial barrier can be estimated from the time course for the accumulation or depletion of a solute at the membrane surface. In 1985 we reported an unstirred layer thickness of approximately 70 microns for Necturus gallbladder epithelium. In our earlier studies the delay caused by noninstantaneous bulk solution mixing was not taken into account and thus the USL thickness was systematically overestimated. In the present studies we describe an analysis of the time course of solute arrival at the membrane surface that takes into account noninstantaneous bulk solution mixing. We also describe a simple technique to monitor the accumulation or depletion of a solute at the membrane surface. The time course for the change in the concentration of either tetramethylammonium (TMA+) or tetrabutylammonium (TBA+) upon elevation of bulk solution concentration is sensed at the membrane surface with an ion-sensitive microelectrode. Because of the high selectivity of the ion-sensitive resin for TMA+ or TBA+ over other monovalent cations in the solution (Na+ and K+), a low concentration (1-2 mM) of the probe can be used. By measuring the time course of the arrival of first one probe and then the other, under identical superfusion conditions, sufficient information is obtained to eliminate multiple fits to the data, obtained when only one probe is used. Neglecting bulk solution mixing caused an error greater than 50% in estimated apparent USL thickness. The effective thickness of the USL depends critically upon chamber geometry, flow rate, and the position of superfusion and suction pipettes. Under our experimental conditions the effective USL at the mucosal surface of Necturus gallbladder epithelium was approximately 40 microns.

Animals↗
PubMed1989

Effects of barium and bicarbonate on glial cells of Necturus optic nerve. Studies with microelectrodes and voltage-sensitive dyes.

We have studied the effects of Ba++, a known K+ channel blocker, on the electrophysiological properties of the glial cells of Necturus optic nerve. The addition of Ba++ reversibly depolarized glial cells by 25-50 mV; the half maximal deplorization was obtained with a Ba++ concentration of approximately 0.3 mM. In the presence of Ba++, the sensitivity of the membrane to changes in K+ was reduced and there was evidence of competition between K+ and Ba++ for the K+ channel. These effects, which were accompanied by a large increase in the input resistance of the glial cells, indicate that Ba++ blocks the K+ conductance in glial cells of Necturus optic nerve. With the K+ conductance reduced, we were able to investigate the presence of other membrane conductances. We found that in the presence of Ba++, the addition of HCO3- caused a Na+-dependent hyperpolarization that was sensitive to the disulfonic stilbene SITS (4-acetamido-4'-isothiocyanostilbene-2, 2'-disulfonic acid). Removal of Na+ resulted in a HCO3- -dependent, SITS-sensitive depolarization. These results are consistent with the presence in the glial membrane of an electrogenic Na+/HCO3- cotransporter in which Na+, HCO3-, and net negative charge are transported in the same direction. In Cl- -free solutions, the Ba++-induced depolarization increased, suggesting a small permeability to Cl-. Using voltage-sensitive dyes and a photodiode array for multiple site optical recording, the distribution of potential changes in response to square pulses of intracellularly injected current were recorded before and after the addition of increased and the decay of amplitude as a function of distance decreased. Such results indicate that Ba++ increases the membrane resistance more than the resistance of the intercellular junctions.

Animals↗
PubMed1991

Cytoplasmic distribution of poly(A)-containing RNA in developing Necturus maculosus oocytes with reference to annulate lamellae.

The cytoplasmic distribution of poly(A)+ mRNA and its relationship to annulate lamellae were examined in developing Necturus maculosus oocytes by in situ hybridization with [3H]poly(U). The specificity of [3H]poly(U) binding was tested by incubating control ovarian sections with either KOH or RNase A before in situ hybridization. In both experiments, the silver grain densities were markedly reduced. Poly(A)+ RNA is uniformly distributed in the cytoplasm until the mid-growth phase and then later in vitellogenesis becomes localized in the subcortical ooplasm. The silver grain density in the cytoplasm varied during oogenesis and was greatest in previtellogenic oocytes. Annulate lamellae commonly are observed with the light microscope in oocytes prior to vitellogenesis. In such oocytes, the labeled mRNA probe is observed over cytoplasmic regions of annulate lamellae. The results suggests that a differential localization of messenger RNA occurs during oogenesis in Necturus maculosus. Furthermore, poly(A)+ RNA is present in cytoplasmic regions of annulate lamellae.

Animals↗
PubMed1984

Organization of interstitial tissue in the testis of the salamander Necturus maculosus (Caudata:Proteidae).

In Necturus maculosus the organization of the interstitial tissue varies according to the stage of spermatogenesis. Leydig cells at various stages of differentiation and myoid cells are always present in this tissue. The Leydig cells are undifferentiated at all phases of germ cell activity and only hypertrophy following spermiation and degeneration of Sertoli cells. These Leydig cells are structurally analogous to mammalian Leydig cells. They do not form part of the lamina propria of the seminiferous lobules and hence cannot be referred to as lobule-boundary cells previously described in the urodele testis (Lofts, '74). When the Leydig cells hypertrophy, numerous unmyelinated axons appear in the interstitial tissue. These axons, often devoid of Schwann-cell cytoplasm, occur in close proximity to Leydig cells. Because the levels of both Substance P and neurotensin increased in the testis of Necturus maculosus as Leydig cells differentiated, we concluded that these neural elements may regulate Leydig-cell function locally, through the release of neuropeptides.

Animals↗
PubMed1984

Fine structure of the abdominal epidermis of the adult mudpuppy, Necturus maculosus (Rafinesque).

The ventral epidermis of adult Necturus maculosus has been studied using electron and light microscopy. Many larval characteristics of amphibian epidermal structure are retained in adult Necturus. The epidermis is a stratified epithelium consisting of four cell layers and five cell types. Major differences compared with other adult amphibians are: the absence of a well defined moulting cycle together with an apparently diminished synthetic and mitotic activity in the stratum germinativum; an outermost cell layer (stratum mucosum) that is unkeratinized and appears to synthesize a mucous layer; and numerous large club-shaped Leydig cells which span the epidermis between the cells of the stratum germinativum and stratum mucosum. The apical region of the stratum granulosum and stratum mucosum cells shows evidence of extensive synthesis. The stratum mucosum appears to be involved in the secretion of vesicular contents onto the outermost surface of the epithelium. The external surfaces of the stratum mucosum cells possess numerous microridges which are supported by an intricate network of cytofilaments in the apical region of these cells. The significance of these features is discussed in relation to the physiology and ecology of this species.

Abdomen↗
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↗
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↗
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↗
PubMed1987

Volume regulation in the early proximal tubule of the Necturus kidney.

The ability of early proximal tubule cells of the Necturus kidney to regulate volume was evaluated using light microscopy, video analysis and conventional microelectrodes. Necturus proximal tubule cells regulate volume in both hyper- and hyposmotic solutions. Volume regulation in hyperosmotic fluids is HCO3- dependent and is associated with a decrease in the relative K+ conductance of the basolateral cell membrane and a decrease in the resistance ratio, Ra/Rbl. Volume regulation in hyposmotic solutions is also dependent upon the presence of HCO3- but is also inhibited by 2 mM Ba2+ in the basolateral solution. Hyposmotic regulation is accompanied by an increase in the relative K+ conductance of the basolateral cell membrane and an increase in Ra/Rbl. Neither hypo- nor hyposmotic regulation have any affect on the depolarization of the basolateral cell membrane potential induced by HCO3- removal. We conclude that volume regulation in the early proximal tubule of the kidney involves both HCO3(-)-dependent transport systems and the baso-lateral K+ conductance.

Animals↗
PubMed1982

Electrical properties of chloride transport across the necturus proximal tubule.

The chloride conductance of the basolateral cell membrane of the Necturus proximal tubule was studied using conventional and chloride-sensitive liquid ion exchange microelectrodes. Individual apical and basolateral cell membrane and shunt resistances, transepithelial and basolateral cell membrane potential differences, and electromotive forces were determined in control and after reductions in extracellular Cl-. When extracellular Cl- activity is reduced in both apical and basolateral solutions the resistance of the shunt increases about 2.8 times over control without any significant change in cell membrane resistances. This suggests a high Cl- conductance of the paracellular shunt but a low Cl- conductance of the cell membranes. Reduction of Cl- in both bathing solutions or only on the basolateral side hyperpolarizes both the basolateral cell membrane potential difference and electromotive force. Hyperpolarization of the basolateral cell membrane potential difference after low Cl- perfusion was abolished by exposure to HCO-3-free solutions and SITS treatment. In control conditions, intracellular Cl- activity was significantly higher than predicted from the equilibrium distribution across both the apical and basolateral cell membranes. Reducing Cl- in only the basolateral solution caused a decreased in intracellular Cl-. From an estimate of the net Cl- flux across the basolateral cell membrane and the electrochemical driving force, a Cl- conductance of the basolateral cell membrane was predicted and compared to measured values. It was concluded that the Cl- conductance of the basolateral cell membrane was not large enough to account for the measured flux of Cl- by electrodiffusion alone. Therefore these results suggest the presence of an electroneutral mechanism for Cl- transport across the basolateral cell membrane of the Necturus proximal tubule cell.

Animals↗