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At least 55 records · Page 3Linked to original sources

PubMed1990

Norepinephrine stimulation of sodium transport in Necturus urinary bladder.

Norepinephrine alters the transepithelial electrical properties of an open-circuited urinary bladder from the mud puppy, Necturus maculosus. When 10(-5) M norepinephrine is superfused over the serosa of the epithelium, the transepithelial voltage (Vt) and short-circuit current (Isc) increase as the resistance (Rt) decreases. The norepinephrine-mediated changes are reversed by the addition of amiloride (5.10(-5) M) to the mucosal Ringer's solution. The serosal adrenoceptors mediating the Na+ transport are more sensitive to norepinephrine (EC50 = 1.2.10(-6) M) than to epinephrine or isoproterenol. Since the Isc is blocked selectively by the antagonist, phenoxybenzamine, stimulation of active transepithelial Na(+)-flux by catecholamines is mediated by an alpha-adrenoceptor. The apical cell membrane voltage (Va) and fractional resistance (fRa) were recorded using conventional KCl-filled microelectrodes. Untreated tissues have Va close to 0 mV while the basolateral membrane voltage (Vb) is between -85 and -95 mV. About 90% of Rt is apical cell membrane resistance (fRa). When amiloride inhibits sodium transport, Va becomes negative, Vb hyperpolarizes slightly and fRa increases to 97%. On the other hand, if the bladders are treated with norepinephrine, fRa decreases to 79% as Va becomes positive and Vb depolarizes. When Rt changes, the resistance of the paracellular pathway (Rp) is unaltered. Changes in the electrical properties of the tissue appear to be mediated primarily by alterations in Ra. Since the Necturus bladder does not respond to antidiuretic hormone, this study implies that biogenic amines regulate Na+ transport in the epithelium.

Animals↗

PubMed1981

Differential distribution of different classes of Necturus retinal ganglion cells.

The responses of Necturus retinal ganglion cells were recorded extracellularly. Each cell was characterized by its response type and the distance between its receptive field center and the center of the optic disc. There was a statistically reliable difference between the locations of sustained-ON cell and the locations of sustained-OFF and ON-OFF cells. Thus, the different classes of Necturus retinal ganglion cells are differentially distributed across the surface of the retina.

Animals↗

PubMed1983

The differentiation of Leydig cells, steroidogenesis, and the spermatogenetic wave in the testis of Necturus maculosus.

The study of seminiferous tubule--Leydig cell interactions in relation to specific germ cell stages during the cycle of the seminiferous epithelium is extremely difficult in most mammalian species due to the continual presence of different spermatogenetic stages in the testis from the onset of puberty. The problem is also compounded by the uniform distribution of both seminiferous tubules and interstitial tissue throughout the entire testis. This difficulty can be circumvented, however, by studying certain species where there is a topographical distribution of germ cell stages within the testis. The urodele amphibian Necturus maculosus exhibits a breeding cycle during which a longitudinal wave of spermatogenesis occurs along the length of the testis, resulting in a spatial and temporal segregation of differentiating germ cells. Moreover, this topographical pattern of spermatogenesis is also reflected in the degree of development of adjacent Leydig cells. This anatomical arrangement allows distinct testicular regions to be obtained using a dissecting microscope. The isolated zones, containing germ cells and Leydig cells in various stages of development, were analyzed for 17 alpha-hydroxylase, C-17,20-lyase, and aromatase activities (key enzymes for the synthesis of androgens and estrogen), estrogen binding, and cytochrome P-450 content. Functional parameters were then correlated with the morphology of Leydig cells in the various zones observed by both light and electron microscopy. It was found that there existed a distinct correlation between the state of differentiation of the leydig cells, their steroidogenic potential, and the distribution of estrogen receptors. These results in Necturus indicate indicate in this species, at least, the steroidal microenvironment of different germ cell associations may be quite specific.

Androgens↗

PubMed1983

Paracellular shunt ultrastructure and changes in fluid transport in Necturus proximal tubule.

To define the structure-function relationships of the lateral intercellular space (LIS), we examined the ultrastructure of Necturus proximal tubule following experimental variation of fluid absorption with or without alteration of active sodium transport. Comparisons were made between (1) control (blood-perfused) kidneys and kidneys doubly perfused using either (2) Ringer solution with organic substrate, (3) Ringer solution with substrate in capillaries but steady-state solution in tubules, (4) Ringer without substrate, or (5) Ringer with low sodium. Intratubular and peritubular capillary pressures were monitored before and during standardized perfusion-fixation for electron microscopy and complete cross-sections of sampled proximal tubules were analyzed by morphometry. All morphometric parameters were the same for proximal tubules in kidneys perfused with blood and kidneys perfused with substrate Ringer. Morphometric parameters of cells and lateral intercellular spaces were the same for tubules with normal volume reabsorption rate (Jv) and with Jv = 0 in substrate-perfused kidneys, and for tubules in kidneys perfused without substrate. However, in kidneys perfused with low sodium, cell height, cell volume, LIS-volume, and average maximum width of LIS were significantly decreased while tight junction length and peritubular length of LIS were unchanged. The results suggest that the measured dimensions of the lateral intercellular space in Necturus proximal tubule are independent of the magnitude of transepithelial salt and fluid movement per se but influenced by hydrostatic pressure gradients and active sodium transport.

Animals↗

PubMed1989

Reconstitution of an inwardly rectifying potassium channel from the basolateral membranes of Necturus enterocytes into planar lipid bilayers.

Basolateral membrane vesicles from Necturus enterocytes, highly (greater than 20-fold) enriched in Na+,K+-ATPase, were reconstituted into planar lipid bilayers. The principal channel activity observed is selective for K+ over Na+ and Cl-. This K+ channel is blocked by Ba2+ and Leiurus quinquestriatus venom but is not affected by Ca2+ over the range of 10(-3) to less than 10(-7) M and is not inhibited by charybdotoxin. L. quinquestriatus venom also markedly reduces the conductance of the basolateral membrane of intact villus cells of Necturus small intestine. The open-time probability (Po) of the channel displays a voltage-dependence characteristic of an "inward rectifier"; i.e., the channel inactivates when the basolateral membrane is depolarized and Po increases with increasing hyperpolarization of that barrier. Assuming that similar properties prevail under physiological conditions, this characteristic could provide, in part, an explanation for the parallelism between Na+-pump and K+-leak activities of the basolateral membrane observed in this epithelium. Thus, an increase in rheogenic Na+-pump activity at the basolateral membrane would hyperpolarize that barrier and, in turn, increase the open time of this K+ channel.

Animals↗

PubMed1992

A peptide from the Drosophila Shaker K+ channel inhibits a voltage-gated K+ channel in basolateral membranes of Necturus enterocytes.

A synthetic peptide composed of the first 22 amino acid residues of the Drosophila Shaker K+ channel inhibits a voltage-gated K+ channel in basolateral membrane vesicles from Necturus enterocytes reconstituted in planar phospholipid bilayers when added to the solution bathing the inner surface of this channel but not when added to the solution bathing its outer surface. A modified peptide in which the leucine in the 7 position is replaced with phenylalanine is also an effective inhibitor, but replacement of the leucine-7 with lysine or glutamate, or digestion with trypsin, renders the peptide ineffective; replacement of the leucine-7 with glycine markedly reduces but does not abolish the effectiveness of the peptide as an inhibitor. These results are analogous to those reported for the Shaker K+ channel +ADHoshi, T., Zagotta, W.N. & Aldrich, R.W. (1990) Science 250, 533-538; and Zagotta, W.N., Hoshi, T. & Aldrich, R.W. (1990) Science 250, 568-571.+BD and suggest that the molecular anatomy of the receptor at the inner face of the Necturus K+ channel with which the peptide interacts to bring about inhibition of that channel may be similar to that of the Shaker K+ channel.

Amino Acid Sequence↗

PubMed1984

Na+-H+ exchange and Na+ entry across the apical membrane of Necturus gallbladder.

The role of Na+-H+ exchange in Na+ transport across the apical membrane was evaluated in Necturus gallbladder epithelium by means of intracellular Na+ activity (aNai) and 22Na+ uptake measurements. Under control conditions, complete replacement of Na+ in the mucosal solution with tetramethylammonium reduced aNai from 14.0 to 6.9 mM in 2 min (P less than 0.001). Mucosal addition of the Na+-H+ exchange inhibitor amiloride (10(-3) M) reduced aNai from 15.0 to 13.3 mM (P less than 0.001), whereas bumetanide (10(-5) and 10(-4) M) had no effect. Na+ influx across the apical membrane was studied by treating the tissues with ouabain, bathing them in Na-free solutions, and suddenly replacing the mucosal solution with an Na-containing solution. When the mucosal solution was replaced with Na-Ringer's, aNai increased at approximately 11 mM/min. This increase was inhibited by 54% by amiloride (10(-3) M, P less than 0.001) and was unaffected by bumetanide (10(-5) M). Amiloride-inhibitable Na+ fluxes across the apical membrane were also induced by the imposition of pH gradients. Na+ influx was also examined in tissues that had not been treated with ouabain. Under control conditions, 22Na+ influx from the mucosal solution into the epithelium was linear over the first 60 s and was inhibited by 40% by amiloride (10(-3) M, P less than 0.001) and by 19% by bumetanide (10(-5) M, P less than 0.025). We conclude that Na+-H+ exchange is a major pathway for Na+ entry in Necturus gallbladder, which accounts for at least half of apical Na+ influx both under transporting conditions and during exposure to ouabain. Bumetanide-inhibitable Na+ entry mechanisms may account for only a smaller fraction of Na+ influx under transporting conditions, and cannot explain influx in ouabain-treated tissues. These results support the hypothesis that NaCl entry results primarily from the operation of parallel Na+-H+ and Cl--HCO-3 exchangers, and not from a bumetanide-inhibitable NaCl cotransporter.

Amiloride↗

PubMed1987

Transcellular sodium fluxes and pump activity in Necturus gall-bladder epithelial cells.

1. Transepithelial Na transport in Necturus was determined by measuring the rate of isotonic fluid flow. The rate at 20 degrees C was equivalent to 175 pmol cm-2 s-1. 2. Ouabain was effective in Necturus, binding to the Na pump in gall-bladder cells with a mean rate constant of 5.4 X 10(3) M-1 s-1. Measurement of the diffusive time constant of the free space for [3H]ouabain shows that the pump must be fully inhibited within 20 s when ouabain is applied to the serosa at 10(-3) M. 3. The serosal Na efflux from loaded cells was inhibited 36% by ouabain equal to a flux of 73 pmol cm-2 s-1. The remaining flux could not be attributed to either exchange diffusion or electrodiffusion induced by ouabain. 4. The transepithelial potential was 0.3 mV serosa positive. The short-circuit current measured was 6.33 +/- 1.9 microA cm-2, equal to a positive univalent ion flux of 65.6 pmol cm-2 s-1 or 38% of the net Na transfer. The current was inhibited within 1-5 min by 5 X 10(-5) M-amiloride. 5. Fluid secretion was immediately inhibited 34% by ouabain, equivalent to an isotonic transport of Na of 59.7 pmol cm-2 s-1. Thereafter it continued for at least an hour, sometimes declining slowly. Amiloride had little effect (13%). 6. The Na pump rate was measured by titrating the cell content with tracer Na at different times after ouabain treatment. The initial slope was equal to a rate of 61.6 pmol cm-2 s-1 or 35% of the net flux at time zero. 7. The Na pump rate has also been measured by recording the rise in cell Na activity with ion-specific micro-electrodes, and correcting for swelling effects. The Na pump rate was very similar to that estimated from the rise in tracer Na content, equal to 59.3 pmol cm-2 s-1 or 31.4% of the transepithelial rate. Examination of the same experiment in the literature shows a closely similar value, about one-third of that expected from fluid secretion or net flux measurements. 8. A scheme is proposed to explain the results, which requires a flow of NaCl through a parallel pathway of small Na content involving exchange en route with the cytoplasmic Na.

Action Potentials↗

PubMed1991

Two types of potassium currents seen in isolated Necturus enterocytes with the single-electrode voltage-clamp technique.

1. The ionic permeability of Necturus maculosus small intestine epithelial cells was investigated using intracellular microelectrodes to measure membrane potential in intact tissue or by the single-electrode voltage-clamp technique in isolated cells. 2. The basolateral membrane of enterocytes appears to be K+ selective as demonstrated by the dependence of membrane potential and fractional serosal resistance measured in the intact epithelium on serosal K+ concentration. 3. Isolated cells had membrane potentials similar to those measured in the intact tissue. Voltage-clamp experiments in a physiological Ringer solution showed the presence of both large outward and inward currents. 4. Removal of Cl- from the bathing medium, linear subtraction or the use of a Cl- channel blocker revealed outwardly rectifying currents. The quasi-linear component was also revealed following K+ channel inhibition; it reversed near ECl, suggesting that the charge carrier was Cl-. 5. Outwardly rectifying currents could be kinetically resolved into two components. A fast component (tau for activation < 4 ms) accounted for 60-80% of the total current at positive potentials. A slowly activating component appeared at voltages positive to 50 mV with tau for activation of > 25 ms. 6. The slow outward current showed strong voltage dependence of both activation and relaxation, which were faster at more depolarized potentials. 7. Both fast and slow outward currents seem to be carried by K+ as they were blocked by Ba2+ and tetraethylammonium (TEA). Tail current analysis of the slow component indicated a reversal potential very similar to EK. 8. Fast outward currents were half-activated at about -40 mV whereas slow outward currents were only apparent at more positive potentials. It is proposed that the fast outward K+ current plays a role, together with Cl- currents, in determining the resting membrane potential of Necturus enterocytes.

Animals↗

PubMed1993

Whole-cell currents in isolated resting Necturus gastric oxynticopeptic cells.

1. Necturus gastric mucosa secretes Cl- actively across the gastric glands which are composed almost entirely of acid- and enzyme-secreting oxynticopeptic cells. Single channel studies on Necturus oxynticopeptic cells have shown that the basolateral membrane possesses multiple K(+)-selective channels but no observable Cl- channels while the apical membrane has Cl- channels but no observable K+ channels. To relate these channel properties to the conductance of the whole cell we have investigated the macroscopic membrane currents with conventional whole-cell patch-clamp techniques. 2. When bathed in amphibian Ringer solution, gastric oxynticopeptic cells had a membrane resistance of 47.8 +/- 2.8 M omega and a membrane capacitance of 75.5 +/- 2.7 pF (n = 82). This gave a specific membrane resistance of 3260 +/- 160 omega cm2 (n = 82). Reversal potentials of the oxynticopeptic cells were -13.8 +/- 1.2 mV (n = 45) for an intracellular Cl- concentration ([Cl-]i) of 42 mM and were significantly more negative -24.4 +/- 3.1 mV (n = 31, P < 0.001) for [Cl-]i = 22 mM. 3. In the absence of ATP in the pipette solution, there was an 80% reduction of the whole-cell current with a typical half-time (t1/2) of 5 min. The run-down was not observed when the pipette solution contained 4 mM ATP. 4. A slow and voltage-independent inhibition of 80% of the whole-cell currents occurred after addition of NPPB (35 microM). Ba2+ (10 mM) produced a reversible inhibition of 20% of the total current. Together, 35 microM NPPB and 10 mM Ba2+ eliminated 95% of the whole-cell currents. These data suggest that in the resting oxynticopeptic cells Cl- carried the major fraction of the current while K+ ions carried only a small fraction. 5. Total replacement of Cl- in the pipette and bath solution by gluconate- increased the membrane resistance to 751 +/- 104 M omega (n = 53) and shifted the reversal potential to -38.1 +/- 2.8 mV (n = 53). 6. Increasing the bath K+ concentration from 6 to 91 mM activated a current which had a high selectivity for K+ over choline+, Li+, Na+, Rb+ and Cs+ and was independent of Cl-. The activation of this K+ current (IK*) by high external K+ was not seen with ATP-free pipette solution. 7. Ba2+ or Cs+ had a voltage-dependent blocking effect of this inward K+ current. Ouabain (1 mM) or SCH 28080 (200 microM), specific inhibitors of the Na+,K(+)-ATPase and H+,K(+)-ATPase, had no effect.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗

PubMed1987

Diphenylamine-2-carboxylate blocks Cl(-)-HCO3- exchange in Necturus gallbladder epithelium.

Intracellular microelectrode techniques were employed to study the effects of diphenylamine-2-carboxylate (DPC) on ion transport in Necturus gallbladder epithelium. Under control conditions, addition of DPC to the mucosal bathing solution caused a concentration-dependent, reversible hyperpolarization of both cell membranes with no measurable resistance changes. In addition, DPC caused the following effects, all consistent with inhibition of apical membrane Cl(-)-HCO3- exchange: fall in intracellular Cl- activity (aCli), increase in intracellular pH (pHi), reduction of the changes in aCli and pHi produced by lowering mucosal solution [Cl-], and reduction of the change in pHi produced by lowering mucosal solution [HCO3-]. Similar studies in theophylline-treated preparations indicate that DPC also inhibits anion exchange under these conditions, but has no effect on the apical membrane electrodiffusive Cl- permeability induced by cyclic AMP. Under these conditions, DPC caused cell membrane hyperpolarization but had no effect on the apparent ratio of membrane resistances. In addition, DPC had no effects on the rapid changes in apical membrane voltage elicited by altering mucosal [Cl-], but caused significant reductions of the slower, secondary voltage changes observed in response to changes in mucosal [Cl-], and the changes in aCli and pHi produced by lowering mucosal [Cl-]. Because others have demonstrated that DPC blocks Cl- channels in other epithelia (Distefano, A., M. Wittner, E. Schlatter, H. J. Lang, H. Englert, and R. Greger. Diphenylamine-2-carboxylate, a blocker of the Cl(-)-conductive pathway in Cl(-)-transporting epithelia. Pfluegers++ Arch. 405: S95-S100, 1985), it is possible that the structures of those channels and that induced by cyclic AMP in Necturus gallbladder are different. Because of its relatively high affinity and rapid reversibility, DPC may become useful in studies of anion exchange in other cells.

Animals↗

PubMed1986

Measurements of intracellular pH in Necturus antral mucosa by microelectrode technique.

Intracellular pH (pHi) was measured in the surface epithelial cells of Necturus antrum using pH-sensitive intracellular microelectrodes. Electrodes were prepared by filling 10- to 20-M omega resistance glass microelectrodes with a H+ ion-selective exchange resin and calibrated before use in solutions of known pH 4.0-8.0. The electrode response (n = 15) was linear (r = 0.93; P less than 0.001) with a slope of 52.1 +/- 2.3 mV/pH unit. Antral mucosa was mounted in a modified Ussing chamber and pHi was determined from the difference between the potentials recorded by intracellular H+-selective and conventional microelectrodes. These measurements of pHi were validated by examining the response of the intracellular microelectrodes to 1) depolarization of the cell membrane produced by K+ substitution for Na+, and 2) alkalinization and acidification of pHi produced by NH+4 substitution for Na+ in the bathing solutions. In tissues bathed with HCO-3-Ringer solution (pH 7.0), the mean pHi was 7.34 +/- 0.02 with a range from 7.24 to 7.43. In N-2-hydroxymethylpiperazine-N' -ethanesulfonic acid (HEPES)-Ringer solution (pH 7.0), pHi was reduced to 7.02 +/- 0.05 (P less than 0.01). Acidification of the luminal solution to pH 6.8 with CO2 produced a 0.22 +/- 0.04-pH unit fall in pHi (P less than 0.001). In contrast, acidification to pH 4.0 with HCl had no significant effects on pHi. These findings indicate that HCO-3 may play an important role in pHi regulation in this tissue. In addition, they suggest that, in contrast to CO2, the surface epithelial cells of Necturus antrum are relatively resistant to acidification by luminal HCl.

Animals↗

PubMed1991

Low doses of ethanol have Ca2+ ionophore-like effects on apical membrane potential of in vitro Necturus antrum.

The effects of low doses of luminal ethanol on the amiloride-sensitive apical membrane potential of Necturus antral mucosa were studied using conventional microelectrode techniques. Luminal ethanol (0.250-4.0% vol/vol) caused a dose-dependent hyperpolarization of the apical membrane potential (Vmc), an increase in transepithelial resistance (Rt) and resistance ratio (Ra/Rb), and a decrease in transepithelial potential (Vms). Luminal amiloride (100 microM) to 4% ethanol-treated antra did not cause any additional hyperpolarization of Vmc. Compared with luminal 2% ethanol-Ringer, an equivalent osmotic mannitol solution depolarized Vmc and basolateral potential (Vcs), decreased Rt and Ra/Rb, and increased Vms. A single dose of 0.50% ethanol attenuated the effects of a second 2% ethanol exposure on Vmc. No change in periodic acid-Schiff (PAS)-positive mucous granule content could be found between control and 2% ethanol-treated antra. The Ca2+ ionophores A23187 or ionomycin (0.25-5.0 microM) dose dependently hyperpolarized the Vmc and Vcs, increased Rt and Ra/Rb, and decreased Vms. Luminal Ca(2+)-free Ringer had no effect on luminal 2.00% ethanol-induced changes in membrane potentials or resistances. Pretreatment with BAPTA blocked by approximately 70 and 55% the Vmc hyperpolarization of 2 and 4% ethanol, respectively. Pretreatment with ruthenium red (10-50 microM) also dose dependently reduced the 2% ethanol-induced changes in Vmc. The data indicate that 1) low doses of luminal ethanol and Ca2+ ionophores have similar effects on Necturus gastric antral membrane potentials and resistances, 2) ethanol-induced hyperpolarizations of the Vmc are partially mediated through an alteration in intracellular Ca2+, and 3) low doses of luminal ethanol do not cause the release of antral epithelial mucous granules at the time when significant changes are occurring in the Vmc.

Animals↗

PubMed1984

Cl- transport via anion exchange in Necturus renal microvillus membranes.

We investigated the mechanism of Cl- transport in microvillus membrane vesicles isolated from Necturus kidneys. Cl- influx was insensitive to changes in membrane potential induced by K+ gradients and the K+ ionophore valinomycin, arguing against conductive Cl- transport. Inward gradients of Na+ or Na+ + K+ did not stimulate initial Cl- influx, arguing against direct Na+-Cl- or Na+-K+-Cl- cotransport. External Cl-, HCO3-, and NO3- each stimulated 36Cl efflux and inhibited 36Cl uptake, indicating anion exchange. Outward HCO3- gradients but not OH- gradients stimulated 36Cl influx, consistent with Cl- -HCO3- exchange. Cl- transport via anion exchange was inhibited by furosemide, bumetanide, and disulfonic stilbenes, but not by acetazolamide. External halides stimulated 36Cl efflux (Cl- = Br- greater than I- greater than F-) but the organic anions lactate, p-aminohippurate, and urate did not. Amiloride-sensitive Na+-H+ exchange was demonstrated. Finally, in the presence of a CO2/HCO3 buffer system, imposing an inward Na+ gradient caused a time-delayed stimulation of 36Cl uptake, consistent with indirect coupling of Na+-H+ and Cl- -HCO3- exchangers. We conclude that the parallel operation of Na+-H+ and Cl- -HCO3- exchangers rather than direct cotransport may account for the Na+-coupled uphill Cl- entry previously observed in the intact proximal tubular cell of Necturus.

Amiloride↗

PubMed1984

Cell and luminal pH in the proximal tubule of Necturus kidney.

Double-barreled, selective microelectrodes filled with liquid ion exchanger were used to determine proximal tubule cell pH (pHcell), luminal pH (pHlum), and peritubular capillary blood pH (pHbl.pt) in Necturus kidney in vivo. The average pHbl.pt of 16 animals was 7.64 +/- 0.3; pHcell was 7.36 +/- 0.02 (n = 50), and pHlum was 7.50 +/- 0.05 (n = 16). Because of the variability in pHbl.pt from one animal to another, we studied the blood/cell/lumen pH differences. We sequentially measured with a single microelectrode pHcell and pHlum, and then pHbl.pt in an adjacent peritubular capillary. In 25 such paired determinations, the average pHbl.pt - pHcell difference was 0.28 +/- 0.03, cell acid, and the pHbl.pt - pHlum difference was 0.14 +/- 0.02, lumen acid. The pHcell in this series was significantly more acid than the pHlum (by 0.14 +/- 0.02), but in a few instances the pH gradient across the apical cell membrane was inversed. All measurements were performed in the initial portion of the proximal tubule. We conclude that 1) proximal cell pH is acid with regard to peritubular blood pH, 2) the proximal tubule of Necturus kidney is capable of establishing a small transepithelial pH difference (lumen acid), and 3) pHcell is generally more acid then pHlum.

Animals↗

PubMed1987

Electrogenic Na/HCO3 cotransport across basolateral membrane of isolated perfused Necturus proximal tubule.

This study was undertaken to determine whether the proximal tubule of the mud puppy Necturus maculosus possesses a basolateral Na/HCO3 cotransporter. We examined the effects on basolateral membrane potential (Vbl) and intracellular pH (pHi) of 1) lowering basolateral [HCO3-] at constant PCO2, and 2) replacing Na+ with N-methyl-D-glucamine. Vbl and pHi were measured with Ling-Gerard and liquid-membrane pH microelectrodes, respectively, in isolated tubules perfused in vitro. We found that decreasing basolateral [HCO3-] from 10 mM (pH 7.5) to 2 mM (pH 6.8) resulted in an immediate depolarization of 14.9 mV, and a pHi decrease of 0.35. SITS (4-acetamido-4'-isothiocyanostibene-2,2'-disulfonic acid, 0.5 mM) inhibited the HCO3-induced depolarization by 87% and inhibited the initial rate of the pHi decrease by 79%. Replacement of basolateral Na+ with N-methyl-D-glucamine resulted in an immediate depolarization of 11.3 mV, and a pHi decrease of 0.36. SITS inhibited the zero Na-induced depolarization by 86% and the initial rate of the pHi decrease by 81%. Nominal removal of basolateral HCO3- (replaced with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) inhibited the zero Na-induced depolarization by 64%, whereas nominal removal of Na+ inhibited the 2 mM HCO3-induced depolarization by 67%. Replacement of all basolateral Cl- with glucuronate did not inhibit the changes in Vbl induced by changing [HCO3-] or [Na+]. Observations similar to those described above have been made previously on Ambystoma proximal tubules, and attributed to an electrogenic Na/HCO3 cotransport mechanism that carries HCO3-, Na+, and net negative charge in the same direction. We conclude that Necturus proximal tubules possess a similar, if not identical, electrogenic Na/HCO3 cotransport mechanism.

Animals↗

PubMed1994

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

1. Taste responses adapt to a constant chemical stimulus. The present study describes a new ionic conductance in taste cells--a Ca(2+)-dependent anion conductance that may explain taste adaptation. 2. Patch-clamp recordings were made on isolated Necturus taste cells or on taste cells in lingual slices. When Na+ and K+ currents were eliminated with tetrodotoxin (TTX) and tetraethyl-ammonium (TEA) in the bath and replacing K+ with N-methyl-D-glucamine (NMDG+) in the pipette, Ca2+ currents were followed by prolonged outward currents. Outward current was abolished when Ca2+ was substituted with Ba2+ or when Cl- was replaced with large organic anions (methanesulfonate, isethionate, or ascorbate). 3. The outward, Ca-dependent current was reduced by certain agents that block Cl- conductances in other tissues, namely 4-acet-amido-4-isothiocyanostilbene-2,2-disulfonic acid (SITS) and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS). However, other Cl- channel blockers--9-AC, furosemide and an antibody to Cl channels-had little or no specific effect on the Ca-dependent outward current in Necturus taste cells. 4. We postulate that the biological action of this Ca-dependent anion conductance in situ is to terminate depolarizing receptor potentials, even during maintained chemostimulation, thereby playing an important role in chemosensory adaptation and modulation of impulse discharge patterns in taste buds.

Afferent Pathways↗

PubMed1985

Control of the heart in the mudpuppy, Necturus maculosus.

The cardiac innervation of the mudpuppy, Necturus maculosus, was investigated using in vivo recording of blood pressure and heart rate, a perfused heart preparation, isolated paced ventricular and atrial strip preparations, Falck-Hillarp fluorescence histochemistry and measurements of plasma concentrations of catecholamines. The heart of Necturus receives a 'classical' vagal inhibitory innervation by cholinergic fibres which act via muscarinic cholinoceptors. In addition, there appears to be an atropine-resistant inhibitory component in the vagal innervation of the heart. This component is best seen at higher frequencies of vagal stimulation (10-20 Hz), but the mechanism behind this response is not known. There is no direct adrenergic innervation of the ventricle or atria, but by use of Falck-Hillarp fluorescence histochemistry, adrenergic fibres have been demonstrated at the atrio-ventricular border, particularly within the intracardiac ganglion. The histochemical evidence is consistent with a modulation of the ganglionic transmission in the vagal pathways by adrenergic neurons and small intensely fluorescent (SIF) cells, but physiological evidence is wanting. An adrenergic control of the heart via circulating adrenaline may occur during 'stress', when the plasma level of adrenaline is high enough to affect cardiac performance.

Acetylcholine↗