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At least 523 records · Page 29Linked to original sources
PubMed1988

Electrogenic Na+/HCO3- cotransport in neuroglia.

Membrane potential recording from glial cells in Necturus optic nerve in the presence of 2 mM Ba++, which was added to block the K+ conductance, gave the following results. 1) In HCO3- -free, low-Na+ solutions (11% of control; Na+ replaced with N-methyl-D-glucamine), the hyperpolarizing effect of adding 10 mM HCO3- was reduced by approximately 80%. 2) 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid (SITS, 0.1 or 1 mM) reduced the effect of HCO3- by at least 50%. 3) In the presence of HCO3-, reduction of Na+ caused a depolarization which was much larger than that observed in nominally HCO3- -free solutions. These observations indicate the presence in the glial membrane of an electrogenic Na+/HCO3- cotransporter in which the stoichiometry of HCO3- to Na+ is greater than 1.

Animals↗
PubMed1996

Effects of serosal-side acidosis on cell pH (pHi) and membrane electrical properties in gastric mucosa.

Acute gastric mucosal injury and bleeding occur in the settings of both respiratory acidosis or metabolic acidosis secondary to systemic sepsis or shock. Respiratory acidosis, however, is more predictably associated with acute injury than metabolic acidosis. We hypothesized that the gastric surface epithelial cells are more susceptible to acute increases in PCO2 than to acute decreases in HCO3-, even for the same level of extracellular acidification. To evaluate this hypothesis, we used intracellular microelectrodes to measure pHi, cell membrane potential (Vc), as well as ion conductances of the apical (Ga) and basolateral (Gb) membranes and the paracellular pathway (Gs) in gastric mucosal cells during acute changes in serosal PCO2 or HCO3-. Necturus antral mucosae were mounted in Ussing chambers, perfused on both sides by Ringer solution (40 mmHg PCO2, 18 mM HCO3-, pH 7.3). Measurements were performed before and during increases in PCO2 (80 mmHg, pH 7.0) or decreases in HCO3- (7.2 mM, pH 6.8 or 2.4 mM, pH 6.4). Both forms of acidosis acidified pHi, depolarized membrane potentials, and decreased ion conductances across apical and basolateral membranes, but not the paracellular pathways. For the same level of extracellular acidification, increases in PCO2 were more effective than acute decreases in HCO3- in acidifying pHi and eliciting disturbances in voltage-generating and ion permeability properties of the cell membranes. These findings suggest that pH-buffering mechanisms in gastric surface cells respond less effectively to high PCO2 than low HCO3.

Acidosis↗
PubMed1986

Is the nuclear envelope a 'generator' of membrane? Developmental sequences in cytomembrane elaboration.

Early diplotene oocytes from Necturus maculosus ranging from approximately 0.2 to 0.5 mm in diameter were examined by electron microscopy. In the smallest oocytes of this range, the cytoplasm is largely devoid of membranes, but contains primarily ribosomes and mitochondria. In slightly larger oocytes, smooth-surfaced cytomembranes first appear in the perinuclear cytoplasm. At this time, the outer layer of the germinal vesicle nuclear envelope (GVNE) shows frequent connections with long membranous lamellae that extend for considerable, but variable distances into the juxtanuclear ooplasm. The number of smooth membranous lamellae increases tremendously as the oocytes increase in diameter. In such oocytes as well, frequent continuities are observed between the outer membrane of the GVNE and many of the cytoplasmic membranes. Eventually, as the ooplasm becomes populated with extensive numbers of membranous lamellae, instances of continuity between the membranous lamellae and nuclear envelope now become sparse and eventually non-existent. The frequent connections observed between membranous lamellae and the outer membrane of the GVNE during a circumscribed interval of diplotene strongly implicate the GVNE in the generation of extensive amounts of cytoplasmic membrane. The ooplasm of larger oocytes in the size range indicated contain numerous Golgi complexes and large quantities of annulate lamellae most of which are positioned in the peripheral or subcortical ooplasm, as well as extensive quantities of smooth membranes of the endoplasmic reticulum and lipid droplets.

Animals↗
PubMed1992

A comparison of intact and in-vitro locomotion in an adult amphibian.

Locomotion was compared in an intact and in-vitro preparation of the adult mudpuppy (Necturus maculatus). The intact animals walked on an aquatic treadmill while in-vitro preparations were made to walk with a bath application of the excitatory amino acid NMA (N-methyl DL-aspartate). EMG recordings of shoulder muscles (pectoralis, latissimus dorsi, dorsalis scapulae, and procoracohumeralis) and elbow muscles (brachialis and extensor ulnae) were obtained from intact animals while recordings were made from only the elbow muscles in-vitro. The in-vitro preparation required magnesium in the bath to initiate and maintain locomotion, consistent with an NMDA mediated response. Also consistent with an NMDA response was the finding that glycine potentiated the NMA induced locomotion in-vitro. The range of cycle durations seen in-vitro was well within the range seen in the intact animal, while gait analysis demonstrated the similarity of intact and in-vitro locomotor cycles. In spite of these very similar locomotor patterns there are interesting differences in the patterned output seen in-vitro, such as the absence of a second burst in the brachialis muscle in-vitro.

Animals↗
PubMed1992

Reconstitution and regulation of an epithelial chloride channel.

We have used a monoclonal antibody (MAb E12), one of several such antibodies raised against theophylline-treated Necturus gallbladder epithelial cells, to isolate a chloride channel protein by the use of an immunoaffinity column and FPLC. This protein (M(r) 219,000) has been reconstituted into a planar lipid bilayer, where it behaves as a chloride-selective channel (PCl/PNa = 20.2; PNa/PK = 1) whose unit conductance is 62.4 +/- 4.6 pS. Antibody added to the trans side (there is no effect from the cis side) causes channel open probability to drop to virtually zero, but has no effect on the conductance or the selectivity of single channels. To test the role of phosphorylation in the activity of the native channel, we studied the effects of the protein phosphatase inhibitor okadaic acid (OA) on intact gallbladders, and showed that channels opened by theophylline treatment and closed by antibody are reopened reversibly by OA (0.01-1.0 microM). Addition of the catalytic subunit of protein phosphatase 2A (PP-2A) to the cis side of a bilayer containing reconstituted chloride channels caused closure of the channels after a delay, and subsequent addition of ATP and the catalytic subunit of cAMP-dependent protein kinase (PKA) caused immediate reopening. These data indicate that (a) this chloride channel protein inserts in a directed way into the bilayer such that the cis side is 'intracellular', (b) the purified channel protein is phosphorylated, and (c) gating from the cellular side is controlled by the direct phosphorylation and dephosphorylation of the channel protein.

Animals↗
PubMed1991

Basolateral electrogenic Na/HCO3 symport in the amphibian distal tubule.

This study was carried out to assess whether the amphibian distal tubule possesses a basolateral Na/(HCO3)n greater than 1 cotransport. The experiments were performed in the kidney of Necturus maculosus in vivo, by means of double-barreled selective microelectrodes. Basolateral membrane potential (Vm), intracellular pH (pHi), intracellular sodium activity (alpha Nai) and intracellular chloride activity (alpha Cli), were recorded during selected disturbances of peritubular fluid composition. The following results were obtained. (a) A sudden decrease of [HCO3]o leads to Vm depolarization, intracellular acidification and decrease of alpha Nai. (b) A rapid fall of [Na]o elicits Vm depolarization and decreases pHi: these patterns are not substantially altered in the presence of millimolar amiloride concentrations or in the nominal absence of peritubular Cl. (c) An acute decrease of [Na]o does not alter alpha Cli. (d) In the functional absence of CO2/HCO3 buffer (nominally CO2-free solution plus methazolamide), the reduction of [Na]o has no effect on Vm and/or pHi. We conclude that the distal tubule basolateral cell membrane is endowed with an electrogenic chloride-independent Na/base carrier, mediating Na and base efflux. The blockade of this carrier by carbonic anhydrase inhibitor indicates that the cotransported base is HCO3 or a related species.

Amiloride↗
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↗
PubMed1992

Thiazide-sensitive Na-Cl cotransport mediates NaCl absorption in amphibian distal tubule.

To find out the mechanism(s) underlying NaCl absorption in the distal tubule of Necturus, we devised a variant of the split-drop technique. Following injection an oil column, subsequently split by a NaCl solution isotonic to plasma, a double-barrelled microelectrode (conventional/selective to Na+ or to Cl- ions) recorded Na+ (alpha Na) or Cl- (alpha Cl) activity and transepithelial potential (Vte). Paired control/low-Na+ solutions yielded reabsorptive half-times (t1/2) of 0.68 +/- 0.11 min and 7.6 +/- 1.8 min respectively; corresponding Vte values were -22.2 +/- 4.0 mV and -7.6 +/- 1.9 mV. t1/2 values of control versus low-Cl- solutions were 0.77 +/- 0.32 min and 6.5 +/- 1.7 min respectively, whereas respective Vte values were not different from one another: -23.8 +/- 4.3 mV versus -18.8 +/- 5.5 mV. Nominally K(+)-free solutions or bumetanide, 10 mumol/l, did not alter t1/2 or Vte, with regard to the paired control. Amiloride, 5 mumol/l or 2 mmol/l, failed to decrease t1/2 or to lower Vte; apparently, the role of a Na+/H+ antiport does not contribute significantly to NaCl absorption. Furosemide, 0.1 mmol/l, reduced t1/2 by 54% with regard to the control state. Determination of t1/2 as a function of increasing hydrochlorothiazide concentrations revealed apical high- and low-affinity sites, estimated at 0.56 mumol/l and 0.115 mmol/l respectively. Taken together these observations indicate that NaCl absorption is predominantly carried out by an electroneutral Na(+)-Cl- cotransport.

Amiloride↗
PubMed1981

The effects of barium on the electrical properties of the basolateral membrane in proximal tubule.

We studied the effects of millimolar Ba2+ concentrations on the properties of the basolateral membrane of the proximal tubule in Necturus kidney. Ba2+ was added in the peritubular perfusate by means of capillary microperfusion experiments. Basolateral membrane p.d. and conductance were continuously monitored in single tubules (their lumen was filled with oil) during reversible application of Ba2+ in peritubular fluid. The effect was membrane depolarization by 14mV and a decrease of membrane conductance to 63% of control values. This association strongly suggests that the main effect of Ba2+ is a decrease of the partial conductance to K+ at the basolateral membrane. However, in other experiments, performed also in oil-filled tubules, Ba2+ was shown to decrease intracellular K+ activity, recorded continuously by means of double-barreled (selective vs non selective) K+ microelectrodes. This and other, indirect evidence support the hypothesis that Ba2+ elicits also a small increase of the partial conductance to sodium at the basolateral membrane.

Animals↗
PubMed1988

Fusion of amphibian proximal convoluted cells into giant cells.

We applied the technique of renal cell fusion to the proximal convoluted tubule of Necturus kidney. Giant fused cells from this segment exhibit stable cell membrane potential, Vm, for several tens of minutes. These cells display a number of electrophysiological properties characteristic of both the apical and basolateral membranes of the proximal tubule. These include 1) significant K+ conductance, 2) no detectable Cl- conductance, 3) apical Na-glucose and Na-amino-acid electrogenic carriers, 4) basolateral ouabain-sensitive Na-K pump activity and 5) basolateral Na/HCO3 cotransport.

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↗
PubMed1987

Effects of membrane potential changes on electrical cell-to-cell coupling in proximal tubule.

Single proximal convoluted tubules (PCT) of Necturus kidney were impaled with three microelectrodes in the sequence M1, M2, M3. M1 was used for injecting short DC current pulses, M2 for recording peritubular membrane potential, V, and M3 for injecting longer DC current steps and thereby shifting V to a new baseline potential, V'. We define the p.d. changes at M2 due to M1-induced pulses as delta V and delta V' (for baselines V and V', respectively). Our objective was to test whether delta V' was equal to delta V. The main finding is that when V depolarized by 10 to 80 mV delta V'/delta V remained close to 1.00. Care was taken to ensure that this apparent stability of the pulse ratio was not due to opposite changes of apical and basolateral membrane conductances (g(A) and g(B) respectively), to changes of the sum g(A)+g(B) compensated for by changes of the cell-to-cell junctional conductance, g(j), or to a distortion of the delta V'/delta V ratio as a function of interelectrode distance, masking voltage-dependent changes of cell membrane conductances. Hyperpolarization of V produced gradual electrical uncoupling between cells as V' became increasingly negative, by a mechanism yet to be determined.

Animals↗
PubMed1986

Intracellular calcium ions as regulators of renal tubular sodium transport.

This review addresses the putative role of intracellular calcium ions in the regulation of sodium transport by renal tubules. Cytoplasmic calcium-ion activities in proximal tubules of Necturus are less than 10(-7) M and can be increased by lowering the electrochemical potential gradient for sodium ions across the peritubular cell membrane, or by addition of quinidine or ionomycin to peritubular fluid. Whereas lowering of the peritubular Na concentration increases cytosolic [Ca++] and [H+], ionomycin, a calcium ionophore, raises intracellular [Ca++] without decreasing pHi. The intracellular calcium-ion level is maintained by transport processes in the plasma membrane and membranes of intracellular organelles, as well as by calcium-binding proteins. Calcium ions inhibit net transport of sodium by reducing the rate of sodium entry across the luminal cell membrane. In the collecting tubule this inhibition is caused, at least in part, by an indirect reduction in the activity of the amiloride-sensitive sodium channel.

Adenosine Triphosphate↗
PubMed1987

Action potentials in epithelial taste receptor cells induced by mucosal calcium.

Chemosensory cells in the taste bud of the tongue of Necturus generate action potentials in response to electrical stimulation through a microelectrode, as recently described by Roper (Science, 220: 1311-1312, 1983). We report that the epithelial receptor cells also respond to 10 mM CaCl2, applied to the mucosal surface, with a depolarization which elicits action potentials when a threshold of -50 mV is reached. Since CaCl2 is one of the taste stimuli in amphibia, the firing of action potentials by chemoreceptor cells may be part of the signal chain in gustatory reception of Ca ions.

Action Potentials↗
PubMed1986

Mechanisms of 1,25(OH)2D3-induced rapid changes of membrane potential in proximal tubule: role of Ca2+-dependent K+ channels.

Eleven different secosteroids or steroids (10(-10) to 10(-8) M) were acutely and reversibly introduced in solutions delivered to the lumen of single proximal tubules of the amphibian Necturus kidney while recording basolateral cell membrane potential Vm. Seven of these molecules (1,25(OH)2D3, 25(OH)D3, 24,25(OH)2D3, 5,6-trans-25(OH)D3, 19-diol-cholesterol, estradiol and testosterone) resulted in changes of Vm (delta Vm) occurring in a few seconds, the largest delta Vm being observed with 1,25(OH)2D3, +6.5 +/- 0.75 mV (n = 19); these seven (seco)steroids, but not the four inactive sterols (vitamin D3, cholesterol, 1 alpha D3 and aldosterone) possess a hydroxyl group on at least one carbon of the C17 to C25 lateral chain of the sterol ring. The delta Vm effect was present in Na+-free or Cl-free media, but it was abolished in HCO3-free media. Depolarization of cell membrane potential by addition of glucose, 11 mM, in luminal perfusion fluid abolished the 1,25(OH)2D3-evoked delta Vm effect, suggesting dependence of the latter on the absolute value of membrane potential. Barium, a blocking agent of K+ conductances, suppressed the 1,25(OH)2D3-evoked delta Vm effect, even when the proper effects of barium of cell membrane potential were canceled by current clamp. Pretreatment with quinine, a putative blocker of Ca2+-dependent K+ channels also abolished the 1,25(OH)2D3-evoked depolarization. Such observations are consistent with the presence of Ca2+-dependent K+ channels at the apical cell membrane of the proximal tubule, these channels being inactivated by 1,25(OH)2D3 and probably by other (seco)steroids.

Animals↗
PubMed1989

Distribution of ion channels on taste cells and its relationship to chemosensory transduction.

The presence and regional localization of voltage-gated ion channels on taste cells in Necturus maculosus were studied. Lingual epithelium was dissected from the animal and placed in a modified Ussing chamber such that individual taste cells could be impaled with intracellular microelectrodes and the chemical environment of the apical and basolateral membranes of cells could be strictly controlled. That is, solutions bathing the mucosal and serosal surfaces of the epithelium could be exchanged independently and the effects of pharmacological agents could be tested selectively on the apical or basolateral membranes of taste cells. In the presence of amphibian physiological saline, action potentials were elicited by passing brief depolarizing current pulses through the recording electrode. Action potentials provided a convenient assay of voltage-gated ion channels. As in other excitable tissues, blocking current through Na+, K+, or Ca2+ channels had predictable and consistent effects on the shape and magnitude of the action potential. A series of experiments was conducted in which the shape and duration of regenerative action potentials were monitored when the ionic composition was altered and/or pharmacological blocking agents were added to the mucosal or to the serosal chamber. We have found the following: (i) voltage-gated K+ channels (delayed rectifier) are found predominately, if not exclusively, on the chemoreceptive apical membrane; (ii) voltage-gated Na+ and Ca2+ channels are found on the apical (chemoreceptive) and basolateral (synaptic) membranes; (iii) there is a K+ leak channel on the basolateral membrane which appears to vary seasonally in its sensitivity to TEA. The nonuniform distribution of voltage-gated K+ channels and their predominance on the apical membrane may be important in taste transduction: alterations in apical K+ conductance may underlie receptor potentials ellicted by rapid stimuli.

Animals↗
PubMed1983

Influx mechanisms for Na+ and Cl- across the brush border membrane of leaky epithelia: a model and microelectrode study.

This paper presents a numerical model for the movement of Na+, K+, Cl-, H+ and HCO3- in a leaky epithelium. The model describes the active transport of Na+ and K+ at the serosal membrane and electrodiffusive permeation across the mucosal, serosal and junctional pathways. The model accounts for H+ and HCO3- production in the cell. The influx of Na+ and Cl- is assumed to occur mainly via Na/H and Cl/HCO3 exchange. The behavior of the cell, with this influx mechanism, is compared to a cell with an obligatory neutral coupled influx of Na+ and Cl-. All parameters are obtained from the literature, primarily from studies utilizing the Necturus gallbladder. The analysis shows (i) that it is virtually impossible in steady-state experiments to distinguish between cells with Na/H - HCO3/Cl transport and cells with Na/Cl transport mechanisms. (ii) That nonsteady-state experiments can decide whether Na/H - HCO3/Cl or Na/Cl transport mechanisms mediate the influx of salt. A comparison between studies with ion-selective microelectrodes and the model predictions indicates that the influx of Na+ and Cl- is mediated by Na/H - HCO3/Cl transport when the external solutions contain CO2 and HCO3. (iii) The model also explains the diuretic effects of furosemide and carbonic anhydrase inhibitor, as well as the stimulatory effects on salt transport of elevated levels of HCO3- at a constant pH. (iv) The model fails to explain some experiments performed in HCO3/Co2-free media and some experiments using inhibitors.

Animals↗